AN ABSTRACT OF THE THESIS OF

Ronald Gibson Coffey for the Ph. D. 1n Biochemi~try_ (Name) (Degree) Date thesis is presented September 6, 1963

Title PATHWAYS OF GLUCOSE UTILIZATION AND RIBOSE

(Major profess or)

14 The production of c o metabolized from glucose labeled in 2 specific carbon atoms by homogena.tes of chick embryonic and adult heart was compared by the radiore spirometric technique. Homogr:­ nates of early (two to five days of incubation) embryo hearts were found to utilize the phosphogluconate pathway of glucose catabolis1n to a greater extent, relative to the glycolytic-Krebs cycle pathway, than did homogenates of hearts from older embryos or adult chicks.

This observation was supported by results with 6- gluconolac­ 14 14 tone -l-C , which was metabolized to to a grea.te r extent than c o 2 14 glucose- U -C by early chick embryo heart. Further, the stimula­ 14 14 tion of the oxidation of glucose-l-c to c by added triphospho­ o 2 pyridine nucleotide was much greater in the early embryo heart than in older embryo or adult heart.

Chick embryos of the ll to 13 s amite stage were explanted for one 14 totwodaysontoamediumcontainingglucose-1, -2, or-6-C , acetat<:;­ 14 14 l-C1~ pyruvate -l-C , ribose -l or ribose- U -C The major chemi­ cal fractions, as well as the degradation products of ribonucleic acid and deoxyribonucleic acid, were examined for radioactivity.

Distinct differences 1n the embryo C!rea and the membrane area were fou_nd Hl th•:' utlhzat1on ot the' labeled substrates. The membrane area incorporated labeled glucose first, followed by a transfer to the 14 emb ryo area. Collectl-on C)f- c 0 at 1nte- rvCI 1s f rom exp1ants a d m1n­. 2 14 istered glucose -1, -2, or -6-C 1nd1cated a small contdbution to oxi­ dative metabolism by the phosphogluconate route. A drop 1n interval 14 recovery of C 0 after 24 hours of explantation was not reflected by C! 2 14 plateau in incorporation of c into the chick embryo. and was inter­ preted as a reduction in the oxidative and an increase in the non-oxida­ tive meta.bohsm of glucose at th1s t1me. 14 The dist nbull on of C 1 n nbose of the chick embryo demonstra­ ted that the non-oxidative pathway for its formation predominated in the explants. The relative contnbuhon of non-ox1dat1ve pathways for ribose synthes1s was estimated to be between 50 and 75 percent for embryos explanted for 24 hours. dnd 80 to 90 percent for 48 -hour ex- plants. About 10 percent of the label in ribose was estimated to have been derived from glucose carbon after conversion to non-pentose cycle or non-glycolyhc pathway intermediates.

Labeling of deoxyribonucleic acid_ as well as the purines and deoxyribose moieties, exceeded that of the nbonucleic acid and the 14 corresponding breakdown products. when glucose, acetate-1-C , or 14 pyruvate-1-C were the substrates but was less than that of ribonu­ 14 - 14 cle1c acid and 1ts constJtuents when nbose -1-C or nbose- U -C were the substrates. The labeling of punnes and pyrimidines of ribonucleic acid and of purines of deoxynbonucleic ac1d suggested 14 that C 0) fixahon was an lmpc_,rL:;nt factor in their formation_ The .(..,

~:Jossibility of ::;eparate pauLo of nL,unucleJc aCJd and deoxyribonucleic 14 acid precursors was suggested by then relatlve labeling from the c substrates. It is suggested thdt the deoxynbose of deoxyribonucleic acid was formed in part by reactions other than the direct reduct1on of ribose. PATHWAYS OF GLUCOSE UTILIZATION AND RIBOSE

SYNTHESIS IN THE CHICK EMBRYO

by

RONALD GIBSON COFFEY

A THESIS

submitted to

OREGON STATE UNIVERSITY

in partial fulfillment of

the requirements for the

degree of

DOCTOR OF PHILOSOPHY

June 1964 APPROVED:

Professor of Chem1stry

In Charge of Major

Head of Department of Chemistry

Dean of Graduate School

Date thesis is presented September 6, 1963 ---- Typed by Betty Thornton ACKNOWLEDGMENT

The Author wishes to express his sincere gratitude to Dr. Robert

W. Newburgh and Dean Vernon H. Cheldelin for their selection of a most interesting research problem, and for their continued interest and guidance in the work.

In addition, he wishes to express gratitude to Alice J. H. R.

Clark for her invaluable aid in the preparation of the chick embryo explants.

Further, he thanks his wlfe for her sustained encouragement in the effort. TABLE OF CONTENTS

Page

Introduction...... l Mate rials...... ll Fertile Eggs ...... ll Adult Chicken Hearts. ll Chemicals ...... ll Homogenate Experiments...... 13 Methods ...... 13 Preparation of Heart Homogenates ...... 13 Radiorespirometric and Warburg Manometric Medium 13 Collection and Counting of c 14o2 ...... 14 Results ...... 15 Discussion...... 27 Summary...... 32 Embryo Explant Experiments...... 33 Methods ...... 33 Embryo Explant Med1um...... 33 Explanation...... 34 Removal and Trimming of Explants ...... 35 Chemical Fractionatl on of Explant Mate rial...... 35 Separation and Degradation of Ribonucleotides ...... 38 Degradation of DNA...... 41 Paper Chromatography...... 42 Degradation of Ribose...... 44 Chemical Determinations...... 45 Results ...... 46 Discussion...... 80 Summary...... 108 Bibliography...... 112 LIST OF TABLES

Table Page

I Effects of ATP, DPN, and TPN on Glucose Oxidation 16

II G :G Ratio for Chick Embryo and Adult Preparations 22 1 6 14 . 14 14 III C 0 Y1elds from ~Gluconolactone-1-C , G-1-C , and2c-u-cl4 Metabolized by Chick Embryo Heart Homogenates...... 28

IV Oxidation of Glucose, Ribose, 6-Phosphogluconate, and 6 -Gluconolactone by Adult Chick Heart Homo­ genates...... 2 9

v Contents of Various Fractions of Chick Embryo Ex- plants...... 51 14 VI Percent c Found in Various Fractions From La­ beled Glucose in Two-Day Chick Embryo Explants 52

VII Specific Activities Found in Various Fractions from Labeled Glucose in One and Two-Day Chick Embryo Explants...... 54

VIII Specific Activities Found in Hydrolysis Products of RNA and DNA Isolated From One and Two-Day Chick Embryo Explants Administered Labeled Glucose: Embryo...... 58

IX Specific Activities Found in Hydrolysis Products of RNA and DNA Isolated From One and Two-Day Chick Embryo Explants ~ Administered Labeled Glucose: Membrane...... 60

X Ratios of Specific Activities of Various Fractions and Nucleic Acid Hydrolysis Products from La­ beled Glucose in Embryo: Membrane Areas..... 63

XI Ratios of Specific Activities of Various Fractions and Nucleic Acid Hydrolysis Products from Dif­ ferently Labeled Glucose-cl4 Substrates...... 65 14 14 XII Incorporation of c from Ribose -l-C , Ribose-U­ cl4, and Pyruvate-l-cl4 into Two-Day Chick Em­ bryo Explants...... 68

XIII Ratios of Specific Activities of Various Fractions and Nucleic Acid Hydrolysis Products from Ri bose -l-cl4, Ribose-U -cl4, and Pyruvate -1-CI 4. . . 71 LIST OF TABLES (CONTINUED)

Table Page . 14 14 XIV Incorporation of C f rom Acetate-1-C and Glucose- 2-ci4 into One and Two-Day Chick Embryo Explants 74

XV Ratios of Specific Activities of Various Fractions and Nucleic Acid Hydrolysis Products from Acetate-l­ cl4 and Glucose-2-Cl4...... 76 14 XVI Distribution of c in Ribose of RNA...... 79 PATHWAYS OF GLUCOSE UTILIZATION AND

RIBOSE SYNTHESIS IN THE CHICK EMBRYO

INTRODUCTION

The relative contributions of the pentose phosphate pathway and the Embden-Meyerhoff (glycolytic) pathway to the utilization of glu­ cose h ave been investigated in a wide va r iety of organisms and tissues (10, p. 318-324; 30, p. 433-465; 34, p . 71 - 94; 56, p. 174­

190; 138, p. 245-269; 145, p . 841-859).

The final cat abolic products of these two pathways, in conjunc­ tion with the Kreb s (tricarboxylic acid) cycle and the cytochrome system for electron transport, are C0 , H 0, and chemical energy 2 2 in the form of adenosine triphosphate (ATP)!../. Importantdifferences

!._/ The following abbreviations are used in this thesis: A TP Adenosine triphosphate APP Adenosine diphosphate DPN Diphosphorpyridine nucleotide TPN Triphosphopyridine nucleotide TPNH Reduced triphosphopyridine nucleotide RNA Ribonucleic acid DNA Deoxyribonucleic acid G-I-c14 Glucose -l-C14 G-2-Cl4 Glucose-2-cl4 G-6-cl4 Glucose-6-c1 4 G-U -cl4 Glucose-U -C14 R-I-cl4 Ribose-l-cl4 R-u-cl4 Ribose-U-c14 PPO 2, 5 -diphenyloxazole POPOP 1, 4 - bis- 2-(5-phenyloxazolyl)-benzene C-2' and -3' -P Cytidine-2'-and-3'-mixed phosphates U-2' and -3'-P Uridine-2' -and-3'-mixed phosphates TCA Trichloroacetic acid Tris 2-Amino-2-hydroxymethyl propane-1, 3 diol CEH Concentrated egg homogenate RTU Relative time unit DPM Disintegrations p er minute 2 in products other than C0 and H 0, arising from these two pathways 2 2 do occur. Thus, formation of triose by either pathway and of q-keto­ glutarate and oxaloacetate by the Kreb s cycle can give rise to amino acids. Formation of acetyl coenzyme A from tnose can give rise to fatty acids. Formation of pentose phosphate, catalyzed bythe pentose

cycle enzymes, can give rise to nucleic acids 1 The first hydrogen acceptor from glucose metabolized by the glycolytic pathway, fol­ lowed by the Kreb s cycle, is diphosphopyridine nucleotide, whereas in the case of the oxidative pentose phosphate pathway, itis principally triphosphopyridine nucleotide. (84, 85). Reduced DPN will generally be oxidized by the cytochrome system, whereas reduced TPN may be oxidized by DPN via transhydrogenase, or it may be used as a source of hydrogen for biological syntheses of compounds suchas fatty acids, deoxyribose, hemoglobin, and reduced folic acid intermediates. (84,

132). TPN is present chiefly in the reduced form in most biological systems investigated, while DPN is present predominantly in the oxidized form. (56, p. 171-222; 84) .

Consequently, when interpreting the relative contributions of the phosphogluconate and the glycolytic pathways to glucose utiliza­ tion, it is important to consider the needs of the organism or tissue for metabolic products other than energy, C0 , and H 0. 2 2 In actively secreting and rapidly proliferating tissues, charac­ terized by a high rate of protein synthesis, the content of ribonucleic acid and the activity of pentose cycle enzymes are high. (20, p. 266­

295; 61, p. 1-12). It has been reasoned that such systems, requiring 3 rapid formation of ribose and deoxynbose for nucleic acid synthesis, might utilize the more direct phosphogluconate oXIdative route to pen­ tose pho sphate to a greater extent than the somewhat less dl.rect, non­ oxidative reactions of glycolysis and the pentose cycle (61, p. 1-12).

The early chick embryo is a rapidly growing system in which the increases in protein and nucleic acid are exponential relative to time (36, p . 336-364; 77, p . 1-50; 78, p. 413-428; 79, 125, 126).

Solomon found that the RNA:E>NA ratio was much higher in the chick embryo blastoderm and the 10-somite stage than in most other tissues of the chick embryo at later stages (126). He states, "The rap1.d rate of cell division during embryogenesis of the chick may be a dominant factor in its metabolism. The rates of increase of wet weight and protein are not suffic1.ent to keep up with cell division and DNA syn­ thesis; consequently there is an apparent loss of wet weight and pro­ tein per embryonic cell. This state only exists for the first three or four days of incubation, after which the instantaneous rate of increase of DNA becomes less and the cell protein and wet weight slowly in­ crease during later stages of differentiation."

In the chick embryo heart, which begins beating at around the

36th hour of incubation, and which reaches its adult morphological form by the eighth day, the protein and DNA contents become constant at about the eighth day of incubation (78, p. 413-428). The RNA:DNA ratio, however, decreases continually from the fourth day of incuba­ tion to hatching {5). Consequently, one might expect that the relative importance of the phosphogluconate pathway to the utilization of 4

glucose would be greater in the early embryo than in the older

embryo or the adult chick. Such a trend might be particularlynotice­

able in heart tissue. 14 By comparing the yields of c o resulting from the metabolic 2 14 14 oxidation of glucose-l-C and glucose -6-c , Krahl concluded that

the phosphogluconate pathway accounts for muc;:h more glucose oxida­

tion than the glycolyti c-Kreb's cycle pathway in early sea urchin em­

bryos (74). Furthermore, the relative contribution of the phospho­

gluconate pathway decreases rapidly as the sea urchin embryo de­

velopes. Jolley obtained qualitatively similar results in the case of

fetal pig heart, using similar methods (61, p. 29-65). He also dem­

onstrated that the oxidative enzymes of the pentose phosphate pathway

were more active in the earlier fetal heart than the older fetus or

adult (61, p. 26-28).

The assessment of radioactive C0 production from a biological 2 14 14 system, to which G-l-C or G-6-c has been administered, may

provide an indication of the importance of the phosphogluconate route

to the total oxidation of glucose. Equations have been developed by

several workers (30, p. 459; 62, p. 858-868; 64, p. 2165-2177; 65). 14 for estimating the percent of glucose ultimately metabolized to c o2 by these two routes. These equations do not yield direct information

as to the quantitative significance of the oxidative and the non-oxida­

tive routes to the synthesis of the pentoses. The specific activity of 14 ribose arising from G-1, -2, or - 6-C , r elative to that of the adrnin­ istered glucose, will yield such information. In addition, the exact 5 distribuhon of the label in the resultant pentoses gives information as to the extent of randonrization occurr1ng 1n the hexose precursor molecule, as well as the route of pentose formation. Thls random­ ization, occurring chiefly by means of pentose cycle reachons, can also be estimated from 1abehng patterns ]n hexose phosphate inter­ mediates or glycogen (64, p.. 2165- 2177; 146).

Bernstein compared the labeling patterns in the hexose of gly­ cogen and the pentose of RNA from adult chick viscera and skeletal 14 muscl e, !ollowmg administration of c -labeled formate, glycine-2­ 14 14 C , and acetate -1-C (12, p. 317-329). H e found that the ch1ck does not utihze the direct oXl.dative pathway to a signif1cant degree to form the pentose of RNA. He concluded that a C + C mecharusm was 3 2 operative. Horecker and Mehler have inte rpreted h1s findings in the light of more recent knowledge of the p entose cycle enzymes to indi­ cate that a comb1nation of glycolys1s and transketolase and transaldo­ lase r eactions could account for the labehng patterns observed (59, p. 207-274). This non-oxidative route, sometimes called the "rever s e pentose cycle 11 or 11 backdoor" route, seems to operate along with th e phosphogluconate oxidative pathway to form the nbose of RNA in mammalian tissue (13, 54, 55, 68, 90).

The contributions of the pentose phosphate pathway and the gly­ colytic-Kreb's cycle pathway to the utilization of glucose, or of the oxidatlve and non-oxidative routes of pentose formation, in the chick embryo have not been reported. The unincubated h en's egg consists of about one p e rcent carbohydrate by weight(115, p. 27). In the yolk, 6 which we1ghs about 19 g., 0. 2 grams of carbohydrate were found, of which about 70 percent was in the form of free glucose. The remalll­ der of the carbohydrate is bound to prote1ns and hp1ds. In the albu­ men, which weighs about 33 g. , 0 . 3 g. of carbohydrate were found, nearly all of which was bound to proteins. Glucose was the only free carbohydrate which could b e detected in the albumen by paper chro­ matography (39, p. 77 -91). Needham demonstrated that carbohydrate is the principal energy source for early chick embryonic development

(97, p. 1165-1184; 98, p.l185-1209; 99, p. 1210-1254). Klein con­ 14 firmed this finding by assessing the produced from embryos c o 2 in intact eggs, following replacement of the yolk or albumen by media 14 containing c -1abeled glucose, amino acids, fatty ac1ds, or proteins

(69, p. 74-81).

Before estimating relative pathways by means of radiotracers, it is desirable to demonstrate the ex1stence of the enzymes and sub­ strates common to the pathways to be considered. Abundant evidence for an active glycolysis in the chick embryo was accumulated by

Needham, although he was unable to demonstrate significant amounts of DPN or substrate level phosphorylation (99, p. 1210-1254). More recent work by Grillo (47), Meyerhof and P erdigon (94), Novikof£ and

Potter and Levy (101, p. 239-252), and Levy and Young (80) showed that phosphorylating glycolysis is indeed active, and that sufficient

DPN is present, in the earliest embryos investigated, to account for the glycolytic rates observed. Hexose phosphate isomerase (112}, fructose -1, 6 -diphosphate aldolase (32, 113), tnose phosphate 7 isomerase (112), and enolase (112) act1v1oes hct ve been measured in early embryos. Numerous workers have compared the actiV1b.es of lactic dehydrogenase at different ages and between different organs of the chlck embryo (89, p . ?70-782, ll3, p. 115-129; 128; 129, p.

182-198; 131). The phosphorylated glycolytlc inte rmed1ates, glucose-

1-phosphate, phosphoglyceric acid, as well as adenosine diphosphate were demonstrated in six to 10-day embryos by Stumpf (133).

Novikoff, Potter and LePage. demonstrated that the intermediates of the glycolytic pathway were present in concentrations comparable to that of adult tissue (101, p. 239-252).

Newburgh, Buckingham, and Herrmann compared the changes in the activities of two TPNH-generating enzymes, glucose-6-phos­ phate dehydrogenase and isocitnc dehydrogenase, 1n whole ch1ck em­ bryos, with respect to changes m protem and DNA (100). Their re­ sults indicatedthat the level of glucose-6-phosphate dehydrogenase per cell decreased during the stage 11 to 17 period (Hamburger and

Hamilton Stages, reference 48, p. 54-67) while the level of isocitric dehydrogenase did not. Glucose -6-phosphate dehydrogenase was measured in chick embryo brain by Burt and Wenger, who found

peaks in activity at about stages 22 and 29 and a minimum at stage 25

(26, p. 87).

Cazorla and Barron measured act1viues of glucose -6-phos­

phate dehydrogenase,isocitric dehydrogenase, glutathione:TPN re­

ductase, and the aldolase-phosphogyceraldehyde dehydrogenase com­

plex in embryos of three to ten days of incubat1on (29). Conversion 8 14 14 . of G-1-C and G-U-C to the nbose of RNA and deoxynbose of DNA was demonstrated m embryonic chick cartllege by Lucy, Webb and

Biggers (86). A combination of both oXJ.datlve and non-oxidab.ve routes of pentose formation was indicated, but no quantitative assess­ ment of the relab.ve contnburions of the two pathways was attempted.

The conversion of ribose and ribose-5-phosphate to lactic acid, and of ribose -5 -phosphate to triose phosphate and to fructose phosphate were measured by Grillo in eight to nine-day chick embryos {46).

Pyruvate oxidase (44; 118, p. 770-782; 131) and all of the Krebs cycle enzymes {5; 32; 44: 100; ll8, p. 770-782; 128; 129, p. 182-198;

131) have been assayed i n the chick embryo. Spratt demonstrated all of the Kreb s cycle oxidauve steps m chlck blastoderms as early as

24 h ours of incubation, as well as in uruncubated eggs, by means of reducing enzyme indicators (131).

The actlvities of the DPNH oxidase and TPNH oxidase systems, as well as diaphorase activity and cytochrome c ox1dase, were measured in chick embryos as ea.rly as two days of incubation by

Brand and Mahler ( 21). Cytochrome c oxidase was measured at 16 hours of incubation by Albaum and Worley (l) and by Moog ~9 4 ). Sipp el measured succinoxidase activity in embryos incubated only 30 hours

(123, p. 205-221). Oxidative phosphorylation was confirmed by Brand and Mahler {21) and by Nowinski and Meyerhoff {102). Brand, Dahl and Mahler measured the concentrations of coenzyme Q- and cyto­ 10 chromes b, c , c, a, and a in mitochondria of 14-day embryo hearts 1 3 and livers (22, p. 2456-2467} . Among lhe many enzymes characterized 9 in the unincubated egg are ovomucoidase, amylase, peptidase, and lipase (81, p. 443-472}. There seems to be little doubt that all of the necessary enzymes are present 1n the ch1ck embryo, from the ear­ liest incubation times yet investigated, for the metabolism of glucose by glycolysis or phosphogluconate pathways, and that the Kreb s c y cle and cytochrome system for oxidative phosphorylation are op­ erative as well. In fact, Moog {95) has stated that 11 No enzyme has yet been shown by sufficiently refined methods to be absent during the first day of incubation. 11

With the knowledge that glucose serves as a principal energy source in the early chick embryo, and that the phosphogluconate pathway is 1mportant in other embryonic systems, 1t seemed desir­ able to investigate the relative contributions of the two predominant routes of conversion of glucose to C0 , as well as the two predom­ 2 inant routes of pentose formation. The work to be reported cons1sts of two parts. The first part entailed collection and measurement of 14 radioactive C0 arising from .-labele d glucose added to homoge­ 2 c nates of heart tissue of embryonic and adult chicks. This approach reaches a practical lower limit at about three days of incubation of the chick embryo, due to the small size of the heart. The second approach made use of the explanting technique first devised by Spratt

(130 , p. 345-365). The embryo explant, growing and differentiating on a nutritive medium containing radioactive glucose, represents a system comparable to an embryo in ~(SO, p. 437 -458; 70; 71; 127) and incorporates significant amounts of labeled glucose carbon into 10 all cellular constituents. 'The explants were used as a source of labeled nucleic acids for degradatJOn studies, and the 1ncorporationof labeled glucose carbon into C0 , protein, ac1d soluble compounds, 2 and ethanol:chloroform soluble compounds was also determined.

Comparisons between the incorporation o£ glucose, ribose, pyruvate and acetate carbon were made. 11

MATERIALS

FERTILE EGGS

Embryos were obtained from white leghorn eggs, purchased

from Hanson Leghorn Farm, CorvalHs, Oregon, and Hyline 950-A

eggs, purchased from Jenk's Hatchery, Tangent, Oregon. The eggs

were incubated at 37°C to 37. 5°C and 58 to 60 percent humidity in a

Jamesway incubator.

ADULT CHICKEN HEARTS

Hearts were removed f rom adult Leghorn hens, directly after

slaughtering at Mutual Produce Company, Corvallis, Oregon, or from

one to two-day-old chicks.

CHEMICALS

14 14 14 D-glucose-1-C , D-glucose-6-C , D-glucose-U ... C , D­ 14 14 14 ribose-l-C , sodium acetate-l-C , and sodium pyruvate-l-C were 14 purchased from New England Nuclear Corporation. D-glucose-2-C was purchased from New England Nuclear Corporation and from Yolk 14 Radiochemical Company. fl-D-gluconolactone -1-C was purchased 14 from Yolk Radiochemical Company. D-ribose-U-C was purchased from Nuclear-Chicago Corporation.

Cytidine-2'-and-3' -mixed phosphates, uridine-2' -and-3' -mixed phosphates, adenosine triphosphate, diphosphopyridine nucleotide, 12 triphosphcpyridine nucleotide, and reduced tnphosphopyr idine nucleotide were purchased from Sigma Chem1cal Corporation.

Adenosine-2' -and-3'-mixed phos phates a.nd guanosi ne- 2 ' - and-3!­ mixed phosphates were purchased from Mann Chemical Company.

Mn-Cellulose Powder 300 DEAE and 300 G/DEAE were purchased from Machery, Nagel and Company, Duren, Germany. 2-Deoxy-D­ ribose-5 - phosphate was obtained from Dr. Donald L. MacDonald as the di(cyclohexylammonium) salt, and converted to the free phosphate by treatment with Dowex SO(W) at room temperature for 24hours (87~

Uridine nucleosidase was prepared from Fleischman•s yeast by the method of Carter (28) . Lactobacillus plantarum (Lactobacillus pen­ tosus) strain 124-2, ATTC #8041, was purchased from American

Type Culture Collection, Washington, D. C. , and was reared as described by Sakami (118, p. 175-177). 13

HOMOGENATE EXPERIMENTS

METHODS

Preparation of Heart Homogenates

Embryonic chick hearts were d1ssec.ted and placed immediately in an ice-cold homogenizing medium consisting of 0. 25M sucrose or

0.14 M KCl, 0. 001 M disodium versene, and 0. 006 M potassium phosphate, adjusted to pH 7. 4 with KOH. Adult ch1cken hearts were kept 1n ice until minced with a razor blade at 0 to 5°C., then placed in the above homogenizing medium. Fat and connectlve tissue were discarded. Homogen1za.tion was performed wlth the Dounce glass homogenizer (35, p. 103). The degree of homogemzatlon variedfrom two to .four strokes with the loose fitting pestle for embryonic hearts to ten strokes with the loose f1tting pestle for adult hearts, followed by two to four strokes with the tight fitting pestle for adult hearts.

This procedure is expected to provide a homogenous suspension of cens, the majority of which are intact (61, p. l3 -15).

Radiorespirometric and Warburg Manometric Medium

The medium was that used by Jolley (61, p. 17) , modified by the addibon of ATP. One ml. of homogenate was added to 2. 00 ml. of a solution which contained the following components: KCl, 0.14M;

MgC1 , 0. 003 M; disodium versene, 0 . 001 M; cytochrome C , 3xl0- S 2 M; ATP, 0. 004 M; nicotinamide, 0. 06 M , and potassium phosphate or 14

Tris, 0. 02 M, pH 7. 4. Bovine serum alburru.n was added to a final

concentration of one percent in the expenments indicated, for the pur­ pose of stabilizi ng certain enzymes which may be unstable at a low total protein concentration. The antibiotics, streptomycin sulfate and penicillin G, were added to a final concent r a lion of 0. 1 percent each, in the experiments indicated, in order to elimjnate bacterial contam­ ination.

14 Collection and Counting of c o 2

The radiorespirometric method described by Wang, et. al., was 14 used for colle ction of c o at half hourly or hourly intervals (141}. In 2 14 early experiments, was collected in 10 ml. of 2 N C0 -free c o 2 2 14 NaOH, and counted as Bac o 3 on aluminum planchets in a thin mica 14 window Geiger-Muller Counter. In later experiments, c o was col­ 2 lected in 15 ml. of 0. 125 N ethanolic Hyamine hydroxide. A five ml. 14 aliquot of each sample was added to 10 ml. of a solution contain­ c o2 ing 6 g. PPO, and 150 mg. POPOP per liter of toluene. Samples wer e counted in a Tracerlab liquid scintillation counter with photomultiplier voltage set at 1550 volts and a gain of 32, without the overpulse reject, or a Packard Tri-Carb model 314-DC liquid scintillation counter with photomultiplier voltages set at l370 volts and pulse discriminator set at 10 to 100 volts and 100 to co volts in two channels. The reading in the lO to 100 volt channel is essentially independent of minor variations in the quenching effects caused by ethanol and the Hyamine hydroxide 15 14 ( 142). In Warburg manometric experiments, C 0 was collected in 2 14 the center well in 20-perceni NaOH and counted as BaC 0 as des ­ 3 cribed above.

RESULTS

The selection of an homogenizing and reaction medium for ra­

diorespirometric exp e riments. requi r es considerable car e . Basically,

a medium is desired which will provide optimum conditions for metab­

olizing glucose to C0 without altering the pathways from those as­ 2 sumed to be natural.

The basic med1um used 1n these expenments was that of Jolley

(61, p. 17). However, this medium did not sustain the oxidative capa­ city of chick heart homogenates beyond an hour. A study of the effects of adding ATP, DPN, and TPN to the reaction m1xture was performed in the hope of improving the oxidative capacitywithoutaltering the ratio 14 14 of radiochemical yields of co from G-l-C and G-6-c . Data in 2 Table I indicate that the additi on of ATP, DPN, or TPN greatly in­ 14 creases the radiochemical yields of c o and the oxidative capacity 2 14 of the homogenate. The addition of TPN tended to increase the c o 2 14 14 yield from G-1-C more than that from G-6-C . This effect was even more marked in the presence of both TPN and DPN. Wenner and

Weinhouse (143, p. 691-704) estimate d that 0. 001 M TPN shifted the percent pa rticipation of tne pentose cyc~e to glucose catabohsm from

47 to 65 percent in rat liver preparations. Thus, T PN would be an Table L Effects of ATP, DPN, and TPN on Glucose Oxidation

14 ~liters 02 Percent C Substrate Cofactor addition consumed Average recovered Gl :G6 14 as c o2 14 G-l-c none 251 333186 1.5 1. 38!:0. 38 14 G-6-c none 416 1.1 14 G-l-c DPN, 3 ~moles 1284 1259t26 27.9 1.13:t.02 14 G-6-c DPN, 3 ~moles 1283 24.7 14 G-6-c TPN, 3 tJ. moles 1221 123ltl0 35.2 1.54!:0 .01 14 G-6-c TPN, 3 tJ. moles 1241 22.8 14 G-l-c ATP, 20 ~moles 1415 1398±17 29.6 1. 13 !0. 01 14 G-6-6 ATP, 20 tJ. moles 1381 26,2 14 G-l-c DPN, 1.5 tJ. moles; ATP, 10 tJ. moles 1312 1282tl0 31.9 0.97t0.02 14 G-6-c DPN, 1.5 tJ. moles; ATP, 10 ~moles 1252 32,8 14 G-l-c DPN, 1.5 ~moles; TPN, 1.5 tJ. moles 1246 1243:t3 55.1 2.40t0.05 14 G-6-c DPN, 1.5 ~moles; TPN, L5 ~moles 1240 22.9

Warburg Manometric Experiment with adult hen heart homogenate. The contents of each flask were the same as those described in Methods, with the exception of the cofactors, which are indicated, Phosphate buffer, 30 0 C, 3 hours, 17

undesirable stimulant of oxidation. DPN might be equallyundesirable,

in view of its selective requirement for glycolytic reactions and most

of the Kreb 1s cycle reactions, as opposed to its non-participation in

the pentose cycle reactions. Thus, the cho1ce of ATP alone, to en­

hance the oxidation of glucose was made b ecause it should have had 14 14 little direct eifect on the ratio of C 0 evolved from G-1-C and 2 14 G-6-c .

The selection of 12 \-& moles ATP per flask was based on the re ­

sults presented in Figure lA. In this experiment, both oxygen uptake 14 14 and radiochemical yield of c o from G-U -c were measured by 2 Warburg manometric methods at various concentrations of ATP.

Kayne, Taylor, and Alpert (66) determined that total reduced pyridine nucleotides and ATP decreased significantly during 1ncubation of rat liver slices, while no change was observed in oxidized pyridine nu­ cleotides or ADP concentration.

Phosphate buffer is commonly used for homogenate studies, and yet it has been shown to i nhib it activities of such enzymes as triose phosphate isomerase (9), t r a n s aldolase (18), and to activate fuma rase at 0. 006 M to 0. 08 M, while inhibiting the enzyme activity at concen ­ trations above 0. 08 M (91). Tris, another common buffer, has been shown to inhibit the reduction of TPN by glucose-6-phosphate dehydro­ genase in the supernatant fraction of rat liver ( 117). The inhibition could be reversed by addition of TPN. Jolley found that the ratio of 14 14 14 . C 0 recovered from G-1-C to that from G-6-C was shghtly 2 higher with phosphate than with Tris buffer (61, p. 45). The data in 18

Figure 1. Effects of Various Additions on Glucose Oxidation by Adult Chick Heart Homogenates. A. B. '0 cl4o2 "0 .-4 cv ]1800~ cv 6 •.-4 0 :I § Phospha C/) C/) ~ A N60 1400§ 0 31200 .::t Oz N N ...... 4 0 0 (.) C/) Ill .j..l 1100~ 1-< ~ cv cv .j..l .j..l (,) •.-4 •.-4 1.4 ~ ~ cv !\ A-I 2 800 :::1.. 0 15 30 0.0 0 .1 0.2

~ moles ATP Moles per liter buffer per flask

'0 c. cv '0 D. '0 cv .-4 600 5 § 900 cv C/) C/) ..... c t: >' 0 0 (,) N 10 500 (,) 0 N N 600 ..;t 0 0 ~ u C/) I C/) I ~ .j..l cv~ ~ 1400 ~ .j..l 300 cv . •.-4 •.-4 (,) ~ ~ ~cv ~ ~ p.. 0 300 0 0 30 0.0 0.5 1.0 ~moles Glucose per ml. of Homogenate flask per flask

Warburg Manometric experiments. All flask components were the same as those given in Methods, with the exception of the following:

14 0 Figure lA. Substrate, G-U-C • Phosphate buffer, 30 C., three hours, 50 minutes. 14 Figure lB. Substrate, G-U-C • Phosphate buffer experiment: 8 0 ~oles ATP per flask, no DPN, 30 C., three hours. Tris buffer experL~ent: 12 pmoles ATP per flask, no DP~ , 300 C, three hours. Figure lC. Phosphate buffer, 300 C, two hours , 15 minutes. Figure lC. Phosphat~ buffer, 31°C, one hour. 19

Figure lB indicate that the concentration of the buffer affects the oxi­

datlve capacity. Phosphate, from 0 . 006 M to 0. 060 M , caused little

c hange in oxygen consumption, whlle at 0. 2 M , oxygen consumption

decreased. However, Tris improved the oxidative capacity as 1ts con­

centration was increased from 0. 007 M to 0. 2 M. Routinely, either

of these buffers was used at 0. 02 M and pH 7. 4 in the radiorespi r o­

metric experiments.

The concentration of glucose used was 2 ~moles per flask. T h e extent of oxidation of glucose varied inversely with its concentr ation , as shown in F1gure lC. The dependence of the activity on homogenate concentration is also appar ent from Figure lD.

The additlon of inert protein to a homogenate might be desirable m view of the fact that some enzymes lose activity when the total pro­ tein concentration is quite low. Such a condition is inevitable in the homogenates of hearts from very young embryos, due to the small amount of tissue which can be prepared in a reasonable time. Bovine serum albumin is often empl oye d to increase the total p rote1n c oncen­ tration. As shown in Figure 2, the addition of bovine serum a lbumin to homogenates of 15-day embryo hearts did not alter the initial r ate of oxygen consumption significantly, but stabilized the homogenates such that oxidation continued for a longer period of time. The rate of D P N ­ stimulatedoxidationofglucose was greater than that of TPN-stimulated oxidation in the absence of bovine serum albumin. The converse was true when bovine serum albumin was added. This finding might indi­ cate a more pronounced stabilizing effect on TPN-dependent reactions 20 Figure 2 . Effect of Bovine Serum Albumin on Oxygen Consumption.

No Bovine Serum Albumin

'0 eQ) :I (/)c: 0 u N 0

Bovine Serum Albumin

'0 so Q) 5 (/) c: 40 0 u

N 0

(/) 30

Q) ~'"' •.-l ...:I

Warburg Manometric experiments with 15 -day chick embryo heart homo ­ genates. All flask components were the same as those given in Methods, with the exception of the additions, as shown, and the omission of 2 ~moles glucose i n the endogenous flask . Glucose Glucose + DPN Endogenous Glucose + TPN 21 than DPN-dependent reactions. However, the effect is not easily inter­ preted, because recent work has shown that bovine serum albumin binds many molec ules, and thus cannot be considered an inert protein

(38, p. 188-201). Bovine serum albumin was added to the medium in the radiorespirometric experiments where indicated.

The presence of antibiotics in homogenate media may be desir­ able in order to rule out the possibility of substrate metabolism by microorganisms. Penicillin G and streptomycin sulfate were added in the experiments indicated. No significant differences in metabolic patterns or oxidative capacity were noted. 14 The possibility of a quantitatively significant retention of c o 2 in the medium of a radiorespirometric experiment was negated by the results of the following experiment with adult heart homogenate. At the end of the actively oxidizing period, as judged by an accompanying

Warburg manometric flask, 0. 2 ml. of 6 NH SO were tipped into two 2 4 14 of four radiorespirometric vessels from sidearms. The C 0 col­ 2 lected during the next ten minutes was negligible from the acidified 14 homogenates, while the c o2 from the control homogenates was about two percent of the total collected during the entire run. 14 14 14 The yields of arising from G-l-C and G-6-C metabo­ c o 2 lized by embryonic and adult chick homogenates, whole embryo homo­ genates, and intact embryonic hearts are expressed as ratios in Table 14 II. The terms G and G indicate the radiochemical yields of c o 1 6 2 14 arising from G-l-C , respectively (30, p. 458). One relative time unit has been defined by Cheldelin, Wang, and King as ''the time Table II. G :G Ratio f or Chick Embryo and Adult Preparations1c 1 6 G :G at 1 RTU Age Tempera­ Bovine Hours 1 6 in ture in serum Anti­ at 2 ll moles 2 ll moles oc days Buffer albumin biotics 1 RTU Control TPN TPNH Embryo: 2 t 37 Phosphate 2. 5 4 . 95 15.8 3 . 5 t 30 Phosphate 4.0 4 . 81 3 . 5-4.0 37 Phosphate 3.0 2. 56 6 . 72 4 . 0 t 30 Tris 4 , 0 2. 50 13 . 6 4 . 5 t 34 Phosphate 3 . 0 1. 71 19 . 0 5 30 Phosphate 2.0 1. 29 5 t 30 Phosphate 3.0 1.44 6 30 Tris + + 5 . 0 1.12 6 f 30 Tris + + 8 . 0 1.01 7 37 Phosphate 5.0 1.08 7 30 Tris + 3 . 0 1.14 8 . 90 7 30 Tris + 4 . 0 1.00 8 30 Tris + 3.5 1.38 10 37 · Phosphate 3.0 1.51 4 . 34 11 30 Phosphate 2. 0 0.94 12 30 Phosphate 3 . 5 1.16 12 30 Tris + 3.0 1.08 12 $ 30 Tris + 8.0 1.07 14 30 Tris 4 .0 1.04 14 30 Tris 4.0 1. 23

Chick: Hatch 37 Phosphate - 5.0 0 . 79 0.90 Hatch+l day 30 Phosphate 2. 0 0.92 Adult 30 Phosphate 3.0 1.02 Adult 30 Phosphate 3 . 0 0.94 N N Table II. G :G Ratio for Chick Embryo and Adult Preparations.* (Continued) 6 ·-­ 1 G :G at 1 RTU Age Tempera­ Bovine Hours 1 6 in ture in serum Anti­ at 2 1-.1. moles 2 ~J,moles days oc Buffer albumin biotics 1 RTU Control TPN TPHN

Chick: Adult 30 Phosphate 3.0 1.06 Adult 30 Phosphate 3.0 1.06 Adult 30 Phosphate 3.0 1.10 Adult 30 Tris 3.0 1.01

Radiorespirometric experiments, All flask components were the same as those describe d in Methods, with the exceptions of the additions indicated.

"k All preparations are heart homogenates, except where otherwise noted. t Homogenate of whole chick embryo. t Homogenate of chick embryo.heart plus a little surrounding tissue. $ Intact chick embryo hearts.

N w 24 required to complete the in1tial catabohc. reac.l1on with respect to the admmistered substrate" (30; p. 448). The end of the lime unit is 14 usually indicated by the rap1d decrease m 1nterval recovery of C 0 2 from C , C , or C of glucose ( 141}. The value for l RTU in the 3 4 2 experiments reported in Table II vaned from two to f1ve hours. At the end of this time the G :G ratio was much higher in the prepara­ 1 6 tions from two to four-day embryos than in older embryos or adults.

The average G :G ratio for adult hen heart homogenates was 1. 03, 1 6 while the ratio for embryonic hearts was significantly greater than unity in most cases. Ratios similar to those of the adult are seen in homogenates from embryos as early as live to seven days, however, and suggest a changing pattern of glucose metabolism at about this time. The additlon of T P N or TPNH caused a marked increase in the

G :G ratio from embryo preparations, but only a slightincrease from 1 6 a hatching chick preparation. This increase was mainly due to a pro ­ 14 14 nounced increase in the c o yields from G-l-C , as the yields from 2 G-6-c14 were not affected o r only slightly depressed from the control yields.

In the two experiments with intact chick embryonic hearts, the

G :G ratios are similar to those for the heart homogenates of embryos 1 6 of corresponding age, suggesting that the pathways of carbohydrate metabolism have not been altered by homogenization. 14 The kinetics of glucose-C decarboxylation by preparations from representative ages of chick embryo and adult are presented graphically in Figures 3 and 4. These f1gures resemble those for 14 14 Figure 3. c o Recovery From Glucose-c Legend: 2 14 0------o Glucose -1-C ._.Glucose-6 -c14 Four-day whole embryo 30 Seven-day embryo heart 14 homogenates homogenates -- Glucose-2-c ~Glucose-u-c 14 20

10

:>..,_. 10 Q) >0 (.) Q) P::i

0 1 2 3 4

N \.]1 14 14 * Figure 4. C o Recovery Fr9m Glucose-C :>. 2 H ~100 Embryo Heart 14-Day Embryo He art Adult Chick Heart 0 u Homogenate s Homogenates (J) ~ .jJ 75 ~ (J) u H ~50 "0 (J) .jJ ~ 25 § 0 u

Homogenates

Intact hearts

Time (Hours) Time (Hours) Time (Hours) . *For legend, see Figure 3 . 27 microorganisms which utilize the phosphoglucona te and the glycolytic-

Kreb s cycle pathways for the utilization of glucose (30, p. 450 - 451). 14 14 It is appa r ent that the initial rate of arising from G-1 ·-C is c o 2 14 greater than from G - 6-c , a nd that differences in total yields from these two substrates are greatest in the four - day embryo homogenate. 14 4 Intact hearts from 12 - day e mbryos m e tabolized G - l-C and G-6-C to 14 at almost equal rates, as did the homogenates of 12-day hearts, c o 2 but the extent of oxidation by the intact hearts was much less than that by the homogenate during the time period used. 14 14 14 The ratio of C 0 yields from ll-gluconolactone -1-C : G-U -C 2 14 14 and from ll-gluconolactone -l-C : G-l-C are highest in homogenate preparations from early embryos, as seen in Table III. The ratios become smaller as the age of the embryo increases. The oxygen con­ sumption by an adult chick heart homogenate due to added glucose was four to five times greater than that due to ll-gluconolactone and 6­ phosphogluconic acid, as shown i n Table IV. Ribose gave rise to al­ most half as much oxygen consumption as did glucose.

DISCUSSION

14 14 14 The ratios of yields from G - l-C and G-6 - C provide c o 2 strong evidence that the phosphogluconate pathway contributes more to the utilization of glucose in early chick embryo homogenates and early chick embryo heart homogenates than in heart homogenates from older embryos, and that this pathway is minor or insignificant in adult chick heart homogenates. The simila r i ty between intact hearts and homo­ , 14 14 genate hearts in the decarboxylahon of G-1-C and G-6 -C suggests 14 14 14 14 Table III. c Yields from 6-Gluconolactone-l-c , G-l-c , and G-u-c o2 Me tabolized by Chick Embryo Heart Homogenates .

14 Age Bovine Yield of C 0 a t 1 RTU as Ratios of yields 2 14 t.-glucono­ .6 - glucono­ in serum percent of administered c . lactone­ l actone 14 14 14 days albumin t.-Gluconolactone-1-C G-l-c G-U-c l-Cl4: l-Cl4: 14 14 G-l-c G-U-c

4 - 95.2 8.2 19.4 11.3 4.9 7 + 83.4 9.7 20.9 8.6 4.0 8 + 48.9 16.3 20.6 3 . 0 2.4 14 - 81.2 48.1 81.4 1.7 1.00

Radiorespirometric experiments. All flash components were the same as those described in Methods, with the exception of .6-gluconolactone-l-cl4, which was added to two ~moles per flask where indicated.

Tris buffer, 30°~ .

N 00 29

Table IV. Oxidation of Glucose, Ribose, 6-Phospho­ gluconate, and ~Gluconocactone by Adult Chick Heart Homogenates .

Substrate ~ liters 0 consumed* 2 ------r------­ Glucose 470

Ribose 210

6-Phosphogluconate 120

6-Gluconolactone 100 ------·-:-­-­ Warburg Manom~tric experiment. All flask components w~re the same as those descr'ibed in Methods, · with the exception of the substrates . Four ~moles of each sub­ strate were added . Tris buffer, 30°C . , two hours and 45 minutes . * j.L liters 0 consumed with substrate minus !J.liters 0 2 2 by endogenous oxidation. 30

that the homogenates provide a true pH.ture of the r e lative importance

of the two pathways.

The Entner-Douder off palhway of h eterolactic fermentation has

never been obser ved in mammals or in higher pla.nts (30, p . 443), and has not b een r epor ted in avian systems. Participation to glucose

utilization by this pathwa y can r easonably be exclude d from considera­

tion in the chick. e mbryo. In fact, this pa thwa y , ope r ating alone or in

conjunction with the phosphogluconate p a thwa y , would require C-1 of 14 glucose to appear as C 0 b efor e C- 2 of glucose, whereas the reverse 2 was true in a ll the ages of chick. e mbryos investigated. Atte mpts to

demonstrate the glyoxilic acid cycle i n the chick embryo failed (88).

The percents of pathway p~ rticipation were not calculated from the chick heart homogenates for the following reasons:

, (1) The term GT- GT 1 which appears in the denominator of ~he equation for the fraction of the administered glucose routed via the phosphogluconate pathway (30, p . 459) was not d etermine d. The term

GT, , r e presenting the fraction of the l a b e l e d substrate engage d in anabolic processes, could probably b e assum e d to b e negligible, since a homogenate system is principally a catabolic r ather than an anabolic 14 system. Attempts to d e monstrate c incorporation into nucleic acids and protein by homogenates failed.

(2) A correction for the term GT, which represents the total r adioactivity of each substr ate administered (30, p. 458), must be made in cases wher e the total r adiochemical yield for any labeled glucose substrate was l ow. If the system demonstrated a rise in 31 decarboxylation activlty, followed by a drop to neglig1ble activity, and 14 no C 1ncorporat1on. . 1nto. ce11u 1ar constituents. could b e d emonstrat e d , 14 then apparently the low yield would reflect a low utilization of c o 2 glucose. If the correction were not made, then percent pathway par­ ticipation calculations would depend as muc h on the utilization of the 14 14 substrate as on the differences in c o y1elds from G-l-C and 2 14 G-6-C .

(3) No reasonable approach to making the correction could be made in the case of the homogenate studies. 14 Using the ratios of yields as an indication of the relative c o 2 importance of the phosphogluconate pathway to glucose utilization, it is apparent that the contribution of this pathway to glucose catabolism is greater the younger the embryo heart homogenate. It is also ap­ parent that at early incubation times, the heart homogenate differs little from the whole embryo homogenate with respect to glucose cata­ bolic pathways. A rather sharp drop in phosphogluconate pathway participation is indicated at incubation times of five to six days . This age corresponds roughly to that at which the morphological develop­ ment of the heart has been nearly c ompleted (78, p. 413-428).

The supporting evidence for this contention lies with the data for 14 14 14 the C yields from a-gluconolactone-1-C and from G-1-C plus o 2 TPN or TPNH. It is assumed that the increase in the G. :G ratio due 6 to added TPN or TPNH represents a proportional increase in theca ­ pacity of the phosphogluconate pathway. Similarly, it is assumed that 14 14 differences m the rano of yields from a-gluconolactone-1-C : c o 2 32 14 G-l-C represent proportional differences in the activities of the phosphogluconate pathway and the glycolytic-Kreb's cycle pathway. 14 When the yields for 6-gluconolactone-l-C are compared with those 14 14 for G-l-C or G-U -c , the participation of the direct oxidative route in the early embryo heart homogenates is seen to be far more impor ­ tant than in the older heart homogenates. The abrupt change in par­ ticipation by the two pathways of glucose metabolism is not evident 14 from the A-gluconolactone-l-C da t a, but a change between incubation ages of four and 14 days is apparent. This conclusion is also obtc.tined by the comparison of G :G ratios in the presence of added TPN or 1 6 TPNH to the G :G ratios in the absence of added TPN or TPNH. 1 6

SUMMARY

(l) Homogenates of early chick embryos and homogenates of early chick embryonic hearts utilized the phosphogluconate pathway of glucose catabolism to a greater extent, relative to the glycolytic-Kreb s

<;: y c l e pathway, than did homogenates of hearts from older chick em­ bryos or adul t chicks.

( 2) An abrupt drop in the relative participation by the phospho­ gluconate pathway in embryo heart homogenates occurs at about fi=ve.to seven days of incubation.

(3} Heart homogenates from adult chicks catabolize glucose al­ most entir ely by the glycolytic-Krebs cycle pathway, with negligible participation by the phosphogluconate pathway. 33

EMBRYO EXPLANT EXPERIMENTS

METHODS

Embryo Explant Medium

Each embryo was explanted onto one ml. of a semi-solid medium modifie d from that described by H a y esh1 a nd Herrmann, and denoted by them as concentrated egg homogenate (CEH) (50, p. 437-458) . This medium consisted of one part fertile unincubated egg homogenate and one part chick Ringer's solution containing 1. 5 percent agar. It was modified to include antibiotics and labeled substrate which could not be a utoclaved with the salt solutions. The modified CEH consisted of ten parts fertile unincubated egg homogenate, four parts ch1ck Ringer' s solution containing four percent agar, five parts chick Ringer's con­ taining 3. 0 mg. per ml. streptomycin sulfate, 0. 5 mg. per ml. peni­ cillin G:Na+, and one part labeled substrate. The antibiotic-containing

Ringer's solution was sterilized by passage through a millipore filter.

The final concentr ation of agar in the medium w as 0. 8 percent instead of 0. 75 percent as described by Hayeshi and Herrmann.

All radioactive substrates wer e adjusted to the same concentra­ tion prior to an exper iment, a nd were appr oximately 2 mg. and 10 IJ.C per ml. Each embryo was thus expose d to about one-half IJ.C, with the exceptlon of the time course incorporation study and the acetate-l-C14 incorporation experiment. In these two experiments, each embryo was exposed to about one-fourth IJ.C. 34

After thorough mixmg, 1. 0 ml. of the final mixture was pipetted onto each evapor ating dish wlu.ch rested on cotton placed in a petri dish. Before the medium had Jelled 1t was cover ed with a two em. s quare p1ece of s te rile lens paper . The lens pe~per provides a uniform surface upon which the embryo m ay rest, and fac1litates the subsequent r emoval of the embryo from the medium, with as little transfer of me­ dium as possible (70). The medium w as pre pared in a sterile room four to 12 hours before explanting, and stored in closed petri dishes in the sterile room prior to expla.nting.

E x plantation

The embryos were removed f rom the eggs afte r 40 hours of incu­ bation in ovo. Embryos of 11 t o 13 sormtes were selected and pre­ pared as " small trim" explants {23) . The trimming procedure involves the removal of the membrane periphera l to the sinus terminalis, as described by Britt and Herrmann (23). Explants w e r e 1ncubated at 370

C. under an atmosphere of 75 percent air and 25 percent oxygen for the first 24 hours, and 95 percent o?'ygen and five percent air for the second 24 hours. These atmospheric conditions a re recommended by

Klein , McConnell and Riquier for optimum growth and development of 14 the explants (71). Radwactive c o was continually swept from the 2 sealed plastic boxes containing the explants, bubbled through concen­ t r a t ed H SO to remove water. and then bubbled through 75 ml. of 2. 0 2 4 14 N e thanolamine m ethanol. c o samples were taken at four to eight 2 hour intervals. A five ml. aliquot was a dded to 10 ml. of the above 35 sc1nt1llat1on solution and counted m the Packard Tn-Carb scintillation counter.

Removal and Tnmming of Explant&

After ex.planting for 24 or 48 hours the embryo explants were

removed from the lens papers, rinsed 1n three changes of chickRinger's, and trlmmed as shown by the dashed hnes m F1.gure 1 of Hayesh1 and

Herrmann (50, p. 440) , separating the embryo from the extraembry­

onic membrane . Each of these sections of the blastoderm were im­

mediately frozen in tubes 1mmersed in a dry 1ce -acetone bath, and

stored at -20°C. prior to the chenucal fractlonatlon.

Chemical Fractlonation of Explant Matenal

Approximately twenty embryos or membranes were transferred

to a small glass hand-operated homogenizer, fitted with a ground­

glass plunger, with two 0. 25 ml. washes of glass -distilled water.

They were homogenized at 0°C. and transfe rred to a three ml. conical

glass centribuge tube with two 0. 25 ml. water washes. All centrifu­

gations were performed w1th a refngerated Servall Centrifuge, at

8000 RPM and 10 mmutes, using the SS-1 head and adaptors. When an

aliquot of the homogenate was withdrawn for the determ1nation of ra­

dioactlVlty the homogenate was transferred to a 2. 00 ml. volumetric

tube, glass distilled water added to the 2 00 ml. mark, mlXed, and

two 0. 050 ml. aliquots withdrawn for scintlllation counting. All scin­

tillation counting solutions of homogenates and chemical fractions 36 were prepared by adding 0. 125N Hyamme hydrox1de 1n absolute ethanol to a volume of 5. 0 ml. , and 10. 0 ml. of a soluhon of 6. 0 g . PPO and

150 mg. of POPOP per liter of toluene. Hyamme hydrox1de has been found to solub1hze protems, anuno ac1ds, and ma.ny other compounds

(2, 140) . Aqueous samples wh1ch exceeded 0 . 30 ml. were dried prior to the addition of Hyam1ne hydroxide , wh1ch was found to solubilize up to 0. 35 ml. of water m tolu e ne. Samples were countedwith the Packard

Tri-Carb scintillation counter as described above. Samples were routinely checked for counting efficiency by the addition of standard b enzo1c. ac1.d -cl4 . Efflciencies varied from one preparation of samples to another from 40 to 50 percent. but the variatlon of efflciencies among samples prepared identically vaned less than one percent.

Observed counts per minute were directly proport1onal to the amount of material added, regardless of the nature of the matenal.

The remainder of the homogenate was then transferred to a three ml. conical glass centrifuge tube. lee -cold TCA was added to a final concentration of £1 ve percent (w/vL mixe d , and centrifuged. The pre­ cipitate was resuspended i n 1ce- cold five percent TCA a nd reprecipi­

0 tated tw1ce. The combined supernatant portions were frozen at -20 C. and saved as the 11 TCA extract. 11

The precipitate was suspended in 95 percent ethanol, mixed, and centrifuged. The r esulting precipitate was resuspended in 1. 5 ml. of ethanol:chloroform (1:3, v/vL incubated at 70°C. for 15 minutes, and centrifuged. This was repeated once. The combmed 95 percent etha­ nol:chloroform supernatants were stored at -200 C. as the "ethanol: 37 chloroform extract." This extract conta1ns hp1de material (49, p . 12).

Nucle1c acids were extracted from the above res1due by the method of Tyner ( 136, p. 187 -188). Th1s consisted of addmg 0 . 75 ml. of 10 percent NaCl, buffered at pH 7 . 5 by a smc.dl amount of Na co , 2 3 to the above residue, and heating at 100°C. for one hour. After cen­ trifugation, the supernatant was passed through about one em. of tightly packed glass wool in the n.p of a micro funnel into a second three ml. conical glass centrifuge tube. Two ml. of 95 percent etha­ nol were added to the supernatant, allowed to stand for four to eight hours at 0°C., and centrifuged to remove the sod1um nucleates . The residue from the first NaCl treatment was treated again wlth 10 percent

NaCl, <.entrifuged and filtered. The flltratJ.on appeared to be neces ­ sary to exclude small amounts of coagulated prote1n which dld not pack readily in the dense NaCl medium. The second sod1um nucleate solu­ tion was added to the fast sodlum nucleate precipitate. Two ml. of

95 p ercent ethanol were added and the m1xture was allowed to stand another four to eight hours at 0°C. It was then centrifuged, and the ethanol supernatant was discarded. A few such ethanol supernatants were examined for radioactivity, whic.h proved to be negligible.

RNA was then separated from DNA by the Schmidt and

Thannhouser procedure (121}. One-half ml. of 0. 2NKOH was added to the dried sodium nucleate fraction, incubated at 37°C. for 20 hours, cooled, and 0 . 20 ml. of 0 . 50 N cold HC10 added. The pH after this 4 addition was six to seven. This was reduced to pH 2. 5 to 3. 0 with cold 0. 1 N HCl, and the mixture was centrifuged to precipitate DNA 38 and KClO . The prec1pitate was washed wlth one ml. of cold 0 . 003 N 4 HCl and centnfuged. The combined supernatants c.ont auung the mixed 2 and 3' phosphates of nbonucleos1des were brought qu1ckly to pH 7 . 0 w1th NH 0H, and glass d 1stilled water Wcu:> added to a final 4 volume of 2. 00 ml. Two 0 . 050 ml. abquots were then taken for scin­ tillation counting, and two for the determinatl.on of RNA content. The remaining RNA solution was dried in~~ for the subsequent separa­ tion of the nucleotides by thin-layer chromotography.

The DNA residue from the HClO -HCl precipitations was washed 4 with ethanol, dried, and stored at - 20°C. The prot.e1n res1due from the NaCl prec1pitations was treated with 0 . OS N NaOH at 30° to 40°C. for complete solution (two to three hours), then made to 5. 0 ml. with d1stilled water. Aliquots of 0 . 20 to 0 . SO ml. were removed for scm­ tillation counting and for total nitrogen determmation.

Separation and Degradation of Rlbonucleotides

Ribonucleotides were separated by thin-layer c hromotography, using MN-Cellulose Powder 300 G/DEAE, as described by Coffey and

Newburgh (31) . Prior to chromatography, it was found necessary to desalt the ribonucleotides. This was done by absorbing them onto a small column of charcoal (20 mg. of Darco G charcoal (57), previously washed w1th 0 . 1 N HCl and distilled water and then made into a 0. 3 x

0. 6 em. column) from which salts were removed by d1shlled water washes. The nbonucleotides were then quantitatively eluted with five ml. of a solutlon of 95 percent ethanol:NH {s . g . 0 . 90):H 0{5: 2: 3, v/v/v~ 3 2 39

(67). The eluant was dried in vacuo for chromatography. Three plates were prepared by mixing 9. 5 g. of DEAE-Cellulose Powder with 62 ml. of glass distilled H 0 and spread onto eight by 12 inch 2 plates at a th1ckness of about 375 microns, using a spreader apparatus obtained from Desaga, Brinkmann Corporatlon. The glass plates were dried at 50°C. for two hours . Standards were spotted at two ern. from the lower edge of the plate and air dried prior to chromatography.

Each experimental sample was repeatedly spotted onto an individual plate for chromatographic separation in two dimensions. The chro­ rnatagrarns were developed in the first dimension in 0. 005 n. HCl, dried, then developed in the second dimension in isobutyric acid: NH 3 (s. g. 0. 90):H 0 (66: 2:32). The spots were located by ultraviolet light 2 and outlined by use of a thin glass rod. The cellulose surrounding the spot was removed with a razor blade, and then the spot itself was re ­ moved by scraping with a razor blade held at a 30° angle to the glass.

This resulted in a small cylinder of cellulose which was then placed in a three ml. conical glass centrifuge tube. To the tube was added 2. 5 ml. of 0. 01 N HCl. This was thoroughly mixed and centrifuged at full speed in a clinical centrifuge for a few minutes, and the supernatant was decanted through a micro funnel, .using Schleicher and Schuell

Number 589 blue ribbon filter parer, which had been previously washed with distilled water. This was adequate to trap the cellulose

"fines. 11 An alternative successful method was to use a fine grade scintered glass filter, omitting the micro funnel and filter paper. The elution was repeated twice and gave 95 to 100 percent recovery of the nucleotides. 40

The aderune and guanine nucleondes were hydrolyzed in 0. 5 N

HCl at 1000 C. for two hours, and the hydrolysate was passed through a small column of charcoal to absorb the purines. The preparation of the charcoal column was the same as that descnbed above. Ribose was eluted quantttatively with a small volume of distilled water and dried in vacuo. The purines were eluted quantitatively with five ml. of a solution of 95 percent ethanol: NH (s . g., 0 . 90): H 0 (5: 2:3, v/v/v) 3 2 (67) and dried in vacuo.

Cytidylic and uridylic acids were dried in vacuo, dissolved in

0.1 ml. of 0. 06 M sodium bicarbonate:sodlUm carbonate buffer, pH 9.1, and incubated w1th 0. 1 ml. of a one mg. per ml. solution of alkaline phosphatase for eight hours at 37°C. A quantitat1ve dephosphorylation was observed by paper chromatography. The resultant nucleosides were than desalted, using a charcoal column, and dried in vacuo.

Cytidine samples were converted to uridine by the VanSlyke nitrous acid reaction as described by Schlenk (119) . To the dried cyti­ dine sample was added 0. 50 ml. of glass distilled water. This was placed in an ice bath, 0 . 50 ml. of 2. 0 N acetic acid a dde d , and 0. 25 ml. of ice-cold 4. 2 N sodium nitr ite added dropwise with mixing. The solution was allowed to stand at 0°C. for two hours , followed by 24 hours at room temperature. This resulted in a 95 to 100 percent con­ version cyhdine to uridine, as determined by the adsorption at 290 and

260 m~ w1th a Beckman DU spectrophotometer. The uridine samples thus prepared were removed from nitrous acid by absorption on char­ coal, and subsequently eluted as above and dned in vacuo. All uridine 41

samples were then treated with uricline nuc.leostdase according to the

method of Carter (28), which cleaves uridine quantitatively to uracil

and ribose The products of thls reaction were separated by the char ­

coal column as described for the products of the purme nucleotide hydrolysate. Ribose and uracil samples were dned in vacuo.

The dried DNA samples were treated with 1. 0 ml. of 0. 5 M HCl for 30 mmutes at 900 C. This procedure was altered by treatmentwith

0. 1 M HCl for 15 minutes at 70°C. in the time course incorporationand 14 the acetate-l-C incorporation experiments. The alteration was made in view of reports of the destruction of deoxyribose by the former treatment (60, 83). Following the treatment, the volume was brought to 2. 00 ml. with distilled water, and 0. 050 ml. aliquots were removed for scintlllation counting and for DNA determination.

The remainder of the DNA hydrolysate was passed through a charcoal column. Deoxyribose-5-phosphate was recovered in the aqueous effluent, neutralized with 0. 10 N NH 0H, and dried~ vacuo. 4 The r ecovery of deoxyribose-5-phosphate from the charcoal column by e lution with 5. 0 ml. of water was only 60 to 80 percent, in contrast wtth the quantitative recovery of ribose with three to four ml. Adenine, guanine, and the pyrimidine deoxynucleosides were recovered in the ammoniacal ethanol effluent, and dned in vacuo. 42

Paper Chromatography

All nbose and deoxyribose - 5 phosphate samples were punfied

by ascendmg chromatography on Whatman Number 41-H paper which had been washed prev10usly W1th distilled water. Ribose chromato­

grams were developed with n-butanol: ethanol: water (52:32: 16, v/v/v)

(104, p. 63). Standard ribose was spotted at either end o£ the sheet,

and detected by the m-phenelenediamine reagent (16 , p. 179)following development. This permitted location of the expe rimental samples.

After cutting these areas, the paper containing labeled ribose were placed in test tubes and eluted with one 2. 0 ml. portion and two 1. 0 ml portions of water. The eluants were filtered through a fine grade scintered glass funnel to eliminate small bits of cellulose fibre which interfere with the orcinol test. Complete removal of ribose was ob­ tained by this method.

The deo.xynbose-5-phosphate chromatograms were developed with isopropanol: NH (s . g. 0 . 90): water (7:1: 2) (87). Standard deoxy­ 3 ribose -5-phosphate was spotted at either end of the sheet, and located with a diphenylamine spray (25) following d evelopment. Areas con­ taining the samples were eluted by the same method used for ribose.

All purine and pyrimidine samples were purified by ascending chromatogr aphy on Whatman No. 1 paper which had previously been washed w1th distilled water. Isopropanol: 2. 0 N HCl (65: 35, v/v) was used as the mobile phase (16 , p . 285). After development of the chro­ matograms, the compounds were located by ultrav1olet light, and 43

identlf1ed by the pos1tions of the correspond.lng standards. Samples

were eluted w1th one 2. 0 ml. and two 1. 0 ml. portlons of 0. 010 N HCl.

Eluants w e re f1ltered through a fine grade scmtered glass funnel to

eliminate small bits of cellulose flbre which would interfere with the

subsequent ultraVlolet absorption assay. The concentration and purity

of each compound eluted was then determ1ned by measureing the ab­

sorbance on the Beckman DU spectrophotometer at 250, 260, 280, and

316 mil . The reading at 316 m\J. was performed in order to provide a

small blank correction for cellulose impurities. None of the purine or pyrimidine compounds absorb light at this wave length, whereas a typical paper blank gave an absorbance of 0. 005 to 0. 030. Unfiltered blanks sometlmes gave an absorbance as h1gh as 0. 20. Ratlos of ab­ sorbance of the blank at 316 ml-1:280 ml!, at 316 mil: 260 m il , and at

316 mil: 250 ffiiJ. were 0. 40, 0. 30, and 0 . 25, respechvely. Thus, the absorbance of the sample at 316 miJ., divided by the r atio for the ab­ sorbance of the blank at 316 mil to another wave length, gave an ap­ proximate blank which was subtracted from the absorbance of the sample at another wave length. After applying this correction, the purine and pyrimidine samples gave ratios of absorbance at 280 m IJ.:

260 ffiiJ. and 250 m1-1: 260 ffit.L which were very close to, or equal to, those recorded in the literature {144, p . 262) .

In cases where the ribonucleotides wer e not separated by thin layer chromatography, the entire mixture was treated w1th HCl as described for the purine nucleotides. Subsequent desalting and chro­ matography was performed as described for purines and pyrimidines, 44

separating adenine, guarune, C-2' and -3 -P c:md U-2' and -3 1 - P (16,

p. 285)

Chromatograph1c separation of aden1ne and guamne samples

from the DNA hydrolysate was performe d as descnbed above. Al­

though deoxycytimne-5 1 -phosphate and thymldlne-5' -phosphate stand­

ards were clearly separated from one another and from the purines ,

experimental sample s pots ove r lapped. The pynmidine deoxynucleo­

tides were not eluted and determined. Schmidt reported that th e hy­

drolytic treatment used would give mixtures of the pyrim1dine nucleo­

tide 5' -diphosphates and 5'monophosphates (120 , p . 757).

Degradation of Ribose

Samples of ribose from va r ious expenments were pooled and

lyophilized. The degradation was carried out by the method of

Bernstein (ll). as described in detail in the "Handbook of Isotope

Tracer Methods" (118, p . 175-178). The acet1c ac1d samples obta1ned

fro m the fermentation of ribose by L actobacillus pentosu s, r e presenting

C 1-2 of ribose, wer e pu rified by steam distillation followed by Celite

column chromatography (118, p . 118 -120) . The lactic acid samples,

representing C 3-5 of r ibose, were separated from the acetic acid

samples by steam distillation, and purif1ed by ether extraction (118, p .

42-44). Acetic acid samples were degraded by the Schmidt reaction

(118, p . 63-66). Lactic acid samples were degraded by manganese

dioxide to acetaldehyde and C0 (118, p. 46-49} . Acetal dehyde was 2 trapped in bisulfite, and the acetaldehyde bisulf1te was oxid1~e d t o 45

acetic ac1d by chromic acid (118 , p 61) The excess chromic acid was

neutrahzed by SnSO and the Cicenc acid was steam distilled, purified, 4 and degraded as before. All 1nchV1dual carbon sampl es were counted 14 as BaC 0 on alum1num planchets w1th a low background, Wlndowless, 3 gas flow Geiger -Muller counter.

Chemical Determma.tions

Total nitrogen was determined by the micro Kjeldahl method of

Lang (75}. using chromatograph1cally pure D, L- serine as a primary

standard. The absorbance was read with a Beckman Model B spectre­

photome te r at 500 mll .

Total phosphorous was determined by a modif1cation ofthe Fiske-

SubbaRow m ethod (7), using Na HPO as a primary standard. The ab­ 2 4 sorbance was r ead at 830 mll··

RNA and ribose were determi ned by the orcinol method of

M e jbaum (92), with the following modifications: Instead of ferric chlo­

ride, ferric sulfate was present in the orcinol reagent at a concentra­

tion of 0. 48 g . per liter. The addition of a ferric salt other thanferric

chloride was suggested by Brown (24) . The final volume of the reac­

tion m1xture was 1. 4 ml. instead of 6 . 0 ml. The h eating time was ex­

tended from 10 to 20 minutes. Samples were r ead at 667 ml!, using

ribose as a primary standard. The amount of RNA was calculated from the amount of ribose determmed by us1ng the conver sion factor

suggested by M e jbaum (92). 46

DNA and deoxyribose were deternuned by the Disc.he diphenyla­ mine reaction described by Burton ( 27) Samples were read at 600 ffi1..L, using deox:ynbose as a primary s td.Ildard. To obtain values for DNA, measured samples of commercial DNA were treCl ted by the Burton method and the color produc.ed was compared to that of deoxynbose.

It was found that 3. 82 IJ.g. of DNA gave the color intensity equal to that for 1. 00 IJ.g . of deoxyribose.

Purines, pyrimidines, nucleosides and nucleotides were deter­ mined in HCl at pH 2. 0 by their absorbance at 260 m!J., using published extinction coefficients (144, p. 262).

RESULTS

14 14 The tncorporation of c from G-6-C into the chick embryo ex­ plant and into the major chemical fractions of the explant during a 48­ hour period is indicated in Figure 5. The initial rate of incorporation into the membrane area was greater than the embryo area, although 14 after 48 hours the embryo had incorporated as much c as the mem ... brane. Radioactive carbon was incorporated into all of the chemical fractions except the TCA extract at an approximately linear rate in the membrane area. Incorporation into the TCA extract reached a plateau at 12 hours increasing only slightly thereafter. An mcreased rate of incorporabon was observed at about 24 hours for all chemical f rac­ tions in the embryo area. This was most marked 1n the protein frac­ tion. 47

14 Figure 5. Recovery of C in Various Fractions of Chick Embryo Exp1ants.*

"0 Q) ~ ....~ 30,000 60,000 "0 Extraembryonic Membrane 0 H Q) 0 -~ 0 .... tiSc: ~ Q) 0 00 tiS 20,00 40,000 s0 ~ 0 :X: .....c: -Q) Q) c: c: co co 1-< 1-< .0 .0 8 ~ ~ 10,00 20,000 :E 1-< 1-< Q) Q,) 0. 0. :E :E Po. Po. A A 30,00 60,000 "0 Q) Embryo ~ tiS ....0 "0 Q,) c: Legend : -~ H co c: c: 20,00 Homogenate 40,000 Q,) 0 00 .... Protein 0 ~ CH3CH20H:CHCl3 0 tiS TCA Extract p::8 DNA ~ RNA - 0 c: >. .... 1-< .0 g, 10'00 20,000 Jl 1-< .0 1-< Q) J3 0. 1-< :E Q,) Po. 0. A ~ 0 A 0 12 24 36 48 Time (Hours) *Four to seven embryos were taken for each determination. 48

The specific activities of the vanous chemical fractions are shown in Figure 6. In this figure. and in all tables reporting specific activities, the following bases were used; TCA and ethanol:chloroform extracts, disintegrations per minute (DPM) per lolg. of phosphorous: protein, DPM per l-lg . of rutrogen; RNA, DPM per l-lg. of RNA; DNA,

DPM per lolg. of DNA. DPM per 1-Lmole express the specific activities of ribose, deoxyribose, purines, pyrimidines, and pyrimidine deriva­ tives.

Specific activities of the ethanol:chloroform extract from the membrane and protein of both embr yo and membrane increased at a constant rate. In contrast, those of the ethanol:chloroform extract of the embryo and the TCA extract of both embryo and membrane areas actually decreased after 18 hours. These decreases may be due to several reasons. One explanation is that an increased rate of incor­ poration of non-labeled compounds from the nutrient medium, relative to that of glucose, might occur, r esulting in a lower specific activity.

Since the specific activities of the TCA and ethanol: chloroform extracts were expreseed as DPM per W5· phosphorous, a decrease in the ratio of carbon to phosphorous could also account for a decrease in the specific activity.

The specific activity of RNA of the embryo area reached a pla­ teau at about 24 hours, followed by a plateau in that of DNA at about

28 hours. Such a plateau is not apparent in the membrane a r ea, al­ though the rates of increase in specific activities of DNA and RNA de ­ cline after about 24 hours. The plateaus of speclfic activity of nucleic 49 Figure 6. Specific Activities in Various Fractions of Chick Embryo Explants. *

1800

>. 4J ·~> Extraembryonic ·~ 4J (.J Membrane < 1200 (.J ·~ ~ ·~ (.J Cl.l 0. (I)

6300 C"'l T-4u :X: u >. 4J 6 ·~ N > Embryo :X: ·~ 4J 1200 4200 UC"'l (.J < 5 (.J ~ . ·~ 0 4J ~ (.J ..... >. CIS (.J 4J H Cl.l ..-I 4J 0. (I) > X 2100j ~ (.J < (.J ..-I ~ ·~ (.J Q) 0. (I) 0 12 24 36 48 Time (Hours) *Four to seven embryos were taken for each determination. See Figure 5 for legend . 50 acids in the embryo are not reflected in the1r actual amounts, which mcrease during the 24 to 48 hour penod at a rate greater than during the 0 to 24 hour period, as seen m Table V.

The percents of c.dded label recovered in the maJor chemical 14 fractions of 48-hour chick embryo explants from G-1. -2, and -6-c are given in Table Vl. The embryo area incorporated approx1mately 14 one-and-one-half times as muc h c a.s the membrane area, and pro­ tein was the most heavily labeled of all the chemical fractions. More radioact]vlty was recovered in metabolic C0 than in the entire homo­ 2 genate, attesting to the high capacity for oxidlZmg glucose at this early age. This is in agreement with the findmgs of Klein (69. p. 74­

81). Recovery of label from the medium, after removal of the explants, varied from 40 to 60 percent of the added label. From four to eight percent of this amount was acidlabile and could be trapped in base. 14 The decarboxylation rates for G-1, -2, and -6-C are compared in Figure 7. Labeled C0 aris1ng from the three substrates was 2 evolved at simHar rates and total amounts , but the initially higher 14 14 rate of c evolution from G-l-C indicates activity of the phospho­ o 2 14 gluconate pathway. The peaks in c recovery at about 24 hours o 2 may indicate a decline in the oxidative uhlization of glucose, as the embryo explants continue to incorporate label at nearly linear rates for the entire 48-hour period.

The specific actlvities of the major chemical fractions, arising 14 from G-1, -2, and -6-c are presented in Table Vli. DNA samples were more heavily labeled than correspondmg RNA samples. However, 51

Table V. Contents of Various Fracti ons of Chick Embryo Explants.*

Extraembryonic Embryo Membrane Time in hours: ot 24 48 ot 24 48 Fraction: TCA Extract Phosphorous 0 . 9 -­ 5.6 4 . 5 -­ 7 . 3 C~CR2 0H:CHC13 Extract Phosphorous 0.4 -­ 2.4 14.4 -­ 9.8 Protein Nitrogenf 4.2 15 . 0 48.0 92.0 56 . 0 54. 0

RNAf 7.1 21.0 61.0 32.0 43.0 62.0

DNAt 2. 1 8 . 8 27.0 71.0 11.0 19. 0

*Data are given as ~g. per embryo . tll-13 somite embryo. tPersonal communication from Dr . Robert W. Newburgh. 14 Table VI. Percent c Found in Various Fractions From Labeled Glucose in Two -Day Chick Embryo Explants. , ______' 14 14 14 Glucose-1-C Glucose -2-C Glucose-6 -C Fraction Embryo Membrane Total Embryo Membrane Total Embryo Membrane Total ---­ - -·· · ------­

TCA Extract 0.69t , OS 0. ss:!:. 12 L54t.l7 0 . 62:1: . 07 0 . 90 :t. 17 1. 51:!= . 23 0 . 60:: . OS O. S2t . ll 1.42±. 13 CH CH 0H : CHC1 O.S7:t.l7 O.Slt:.11 L6S:t ,25 0 . 99t , 26 0 , 77:1: , 11 1.76±.35 O.S5t:. l8 0.771 .08 1. 62"±. 16 3 2 3 Extract I I Protein 2 . 36t.26 1.09±,22 3 . 46±, 50 L 67!. 23 0 . 7lf . l4 2 . 3S'!. 42 2 . 54±. 47 1. 20t ' 47 3 . 73±. 72 RNA 0,61±".05 0.2S±,02 O, S9±,09 0 , 97 r . 45 0.24±. 02 L21±. 2S 0 . 50±" .11 0 . 26:!: . 09 0.75:±".13 DNA 0.27±.04 0.09±,02 0 ,36 :1: , 05 0,27±,05 O. OS±',02 0 .3 6:!:" , 06 O. lS:t-. 05 0 ., 0S:t .02 0 . 25±' . 05

Total 4 . S3t. 2S 3 , 21±-.47 S . 03±. 60 4 .3S'± , 4S 2.93±". 51 7.3 l±. S6 4 . 65±". 50 3 . 22±". 39 7. SSt-. 91

Original Homogenate 4.94±.25 3.37:t-.22 S . 31±. 44 4 . 59r, 94 3.71±-. Sl S. 30:±-1.63 4 . 15'±Ll7 3 . 13:t-. 69 7 . 2St'l.54

C0 12 . 70 ±L51 lL SO:t0 . 71 10 . 12±LOS 2

Average and standard errors of the mean are given for six experiments . Each determination represents 15 to 20 embryos or membranes. The standard errors of the mean were calculate d according to the formula:

2 OM =[_'2x /N(N-1) ]~ (137, p . 110) where X is the deviation of a determination from the me an, and N is the number of determinations.

(.}1 N 53

14 Figure 7. c Recovered From Chick Embryo Explants o2 Admin~ istered Labeled Glucose.

15

>. 1-< Legend: Cll > 0 14 t) ~ G~l~C Cll 10 0::: c~2~c 14 .w 14 c:: o------e G~6~c Cll t) -- 1-< Cll p.., Cll 5 •>.-l .w tiS .-I s::s (.) t)

>. 1-< Cll ~ 2.0 t) Cll 0::: .w c:: Cll t) 1-< Cll p.., 1.0

00&------~------~------~------~------~ 1 10 20 30 40 so

Time (Hours) 54 Table VII . Spe c i f i c Activities Found in Various Fractions From Labled Glucose i n One and Two Day Chi c k Embryo Explants .

Embryo

14 14 14 Glucose-1-C Glucose-2-C Glucose-6-C DPMlj.tg. OM N DPM ~ug . OM N DPM/flS .

Fraction days TCA Extract 2 1340 ! 100 8 1230 't 130 6 1220 1:. 130 8

TCA Extract* 2 1590 !; 120 8 1460 ! 150 6 1450 ~ 150 8 CH CH 0H:CHC1 3 2 2 Extract 2 3210 ! 630 7 3560 1" 790 6 3410 ! 740 7

Protein 2 479 t 42 7 371 ~ 19 6 513 ~ 18 7

RNA 1 153 ! 15 8 186 t 25 8 165 1. 18 8 RNA* 1 232 .:: 21 8 281 :t 27 8 235 -! 28 8

RNA 2 246 ~ 18 7 271 :t 26 6 266 ! 35 7

RNA* 2 290 ! 26 7 315 :1 29 6 301 ! 40 7 DNA 2 355 !. 35 3 375 t 7 3 312 '! 8 3

DNA* 2 381 -!, 40 3 411 't 7 3 343 ~ 19 3

Extraembryonic Membrane

14 14 14 G1ucose-1-C Glucose-2-C Glucose-6 -C DPM/j.(g. £1}1 N DPM/,ug . OM N DPM/~g . ~ N f.:ii!.s;tiQn days TCA Extract 2 1270 t 90 8 1350 t 130 6 1350 "!. 160 8 TCA Extract* 2 3270 :t 290 8 3450 ! 260 6 3230 1 380 8 CH 0H : CHC1 3cH2 2 Extract 2 812 t 73 7 808 :! 73 6 1010 ! 110 7

Protein 2 283 :! 22 7 223 t 19 6 285 't 13 7

RNA 1 76 :! 9 8 77 ~ 8 7 73 ~ 7 7 RNA* 1 312 -: 35 8 301 ~ 32 7 285 "t 24 8

RNA 2 145 ;! 13 7 146 ~ 12 6 139 '! 11 7

RNA* 2 284 1: 31 7 300 ~ 21 6 285 't 33 7 55 Table VII . Specific Activities Found in Various Fractions From Labled Glucose in One and Two Day Chick Embryo Explants. (Continued)

Extraembryonic Membrane

14' 14 14 Glucose-1-C Glucose-2-C Glucose- 6 - C DPM&tg. OM N DPM/,ug. OM N DPMIJIS· OM N

Fraction days DNA 2 201 t 12 3 236 :l 23 3 301 t 33 3 DNA* 2 319 i 18 3 375 ! 38 3 '411 ! 38 3

Results were averaged and the standard errors of the mean,~ were cal­ culated for the number of determinations N. Each determination represents 15 to 20 embryos or membranes. * The 4 specific activity was calculated as DPM per O)lS· as described in the text. 56 one may calculate a t, specific activity based on the increment of a parameter during the period of exposure to the labeled substrate.

Calculations of DPM per 6 [..tg , were performed for the RNA, DNA, and TCA soluble fractions , usi ng the data in T able Vand the recoverie() obtained. The 6 specific activities permit a better comparison be~ tween incoorporation into embryo and membrane areas, which have quite different contents of material and different rates of synthesis of new material. The calculations were not attempted for the protein samples and the ethanol:chloroform extracts, as the contents of these two fractions in the membrane are greater at zero time than at 48 hours . This is probably due to adhering yolk granules , which cannot be thoroughly washed off the embryo prior to explanting, but which w ould probably diffuse about afte r explanting. A comparison of the 1:1 specific activities of RNA and DNA reveals smaller differences than found with the uncorrected values. The validity of the comparison would depend upon diffusion of the compounds from one locus to another, as well as their degradation or transformation to other compounds.

These processes might be considered more important for the TCA­ soluble compounds than for the large nucleic acid molecules.

The plateaus in the specific activities of RNA and DNA in the embryo area, observed in Figure 6 , are not apparent from the data presented in Table VIL Since many more determinations are repre­ sented in Table VII, it may be concluded that the plateau observed in

Figure 6 represents an experimental error. Although explants which had died or not developed prope rly were discarded, large differences 57

1n specific activities of all chemical param~ters from one experiment

to another were always observed" Muc h smaller differences in ratios

of specific activities of various parameters were found.

The specific activities of nucleic acid hydrolysis products, aris­ 14 ing from G-1, -2, and -6-c , are given in Table VIII for the ~mbryo

area and Table IX for the membrane area. Ribpse samples from one­

day embryo explants taken from the uridine moiety of RNA were more

heavily labeled than those associated with the other bases . Small dif­

ferences were noted in the specific activitie~ of ribose associated with

different bases in the two - day embryo explants. A comparison of

specific activities of ribose and deoxyribose, as well as purines from

RNA and DNA, reveals that all DNA hydrolysis products studied were

labeled more heavily than corresponding RNA hydrolysis products .

The specific activities of deoxyribose taken from the membrane area

of one and two-day embryo explant$ were neal'ly the same, while

those from the embryo area were much greater from the two-day than

the one -day embryo explants. All ribose samples were two to three

times more radioactive in twq-day than in one-day embryo explant:s.

Cytosine and uracil of RNA were more radioactive than adenine

and guanine in the one-day embryo explants, regardless of the labeled

substrate .. In the two-day embryo explants, cytosine and uracil la­ 14 beled from G-1 and -6 -c were more radioactive than correspo;nding

purine samples, wltile small differences were observed when G- z.~c;} 4 was the substrate. Specific activities in RNA pyrimidines arising 14 from G-2- and -6-c are smaller from two-day than from one-dc;ty Table VIII. Speci fic Activities Found in Hydrolysis Products of RNA and DNA Isolated from One and Two Day Chick Embryo Explants Administered Labeled Glucose

Embryo

14 14 14 Glucos e-1-C Glucose-2-C Glucose-6-C DPM DPM DPM

N Compound Source Days l..l mole OM ~ - 1..1. mole ()M N l..lmOl e crM N

Ribose Adenosine RNA 1 10,000 1 12,900 1 12 ,900 1 Ribose Guanosine RNA 1 11,500 1 12,700 1 12,500 1

Ribose Cytidine RNA 1 12,600~5,700 2 15,900±5,900 2 16' 600 ±6' 600 2 Ribose Uridine RNA 1 16,600-=1,900 2 18,600±5,300 2 21, 600 ±-1' 000 2 Ribose Adenosine RNA 2 34,500-±:1,500 7 31,700"!:2,100 6 34,400±1,400 7 Ribose Guanosine RNA 2 30,200"'tl,800 7 32,800-!:1,800 6 35' 600±1' 800 7 Ribose Cytidine RNA 2 29,400t3,000 7 38' 000"!::3 '000 6 33' 000±3' 300 7 Ribose Uridine RNA 2 30,000t3,000 7 37,400t3,100 6 37, 2oo±5, 100 7

Adenine RNA 1 7,000"!:3,500 2 3,800"!:1,300 2 6,500 1 Guanine RNA 1 8,800-=4,800 2 8, 900­+ 500 2 Cytosine RNA 1 23,600"!:3,900 2 24,400"±1,100 2 29, lOO-t 300 2 Uracil RNA 1 19,800tl,300 17,000!6,000 2 21,400!6, 700 2 -­ -----~------z_l 1,]1 00 Table VIII. Specific Activities Found in Hydrolysis Products of RNA and DNA Isolated from One and Two Day Chick Embryo Explants Administered Labeled Glucose (Continued)

Embryo

14 14 14 Glucose - 1-C Glucose-2-C Glucose -6 -C DPM DPM DPM Compound Source Days ''""t-tmole ~ N t-t mole OM N t-t mole (JM N

Adenine RNA 2 13, ooot 400 7 11, 900!: 400 6 11' 500~ 300 7 Guanine RNA 2 10, ooo= 300 7 9,700:tl,400 6 9,500t 500 6 Cytosine RNA 2 28,200tl,200 3 10,900-t 200 3 16' 900"!' 900 3 Uracil RNA 2 27,700-:!:6,700 3 11, 200:! 700 3 17,800! 600 3

Deoxyribose DNA 1 24,800t2, 700 8 35 '000±3' 800 8 34,000t3,600 8 Deoxyribose DNA 2 40,000±7,700 7 45' 200:!5' 600 6 49,600:!:3,900 7

Adenine DNA 2 19,600tl,600 7 18,300i:l,400 6 17 ,400±1, 700 7 Guanine DNA 2 20,100±2,400 7 18,100'11,100 6 17' 700-:tl' 800 7

(JI -.!) Table IX. Specific Activities Found i n Hydrolysis Products of RNA and DNA Isolated from One and Two Day Chick Embr yo Explant s Administered Labeled Glucose

Extraembryonic Membrane

14 14 14 Glucose -1-C Glucose-2-C Glucose-6 -C DPM DPM DPM Compound Source Days ~l mole 6M N 1-L mole OM N 1-L mole <>M N

Ribose Adenosine RNA 1 10,600 1 12,200 1 13' 700 1 Ribose Guanosine RNA 1 9, 700 1 13' 700 1 Ribose Cytidine RNA 1 7,200"!: 800 2 11, 900t 500 2 12,600"t l,500 2 Ribose Uridine RNA 1 13, 700tl,500 2 17,300±3,900 2 14, lOOt 600 2 Ribose Adenosine RNA 2 20,200t2,000 7 22,100±1,400 6 20,600::1: 2,400 7 Ribose Guanosine RNA 2 18,800t. 800 7 21, OOOtl, 900 5 21,900'!::1,300 7 Ribose Cytidine RNA 2 15,700tl,700 7 22, 700tl,500 5 18' 600 ::!:1, 900 7 Ribose Uridine RNA 2 18,100il,800 7 20,600tl,400 6 20,400±1, 700 7

Adenine RNA 1 5,400;tl,200 2 8,900t 500 2 8,900±.2,200 2 Guanine RNA 1 6,500± 900 2 7' 100! 800 2 Cytosine RNA 1 13' 900il, 600 2 10,1001. 200 2 16,400:! 400 2 Uracil RNA 1 15,900"1 800 2 17 ,OOO·tl, 700 2 14,500!2,200 2

Adenine RNA 2 8,800± 200 7 8' 700 t 700 6 7' 600± 500 7 Table IX. Specific Activities Found i n Hydrolysis Products of RNA and DNA Isolated from One and Two Day Chick Embryo Explants Administered Labeled Glucose (Continued)

Extraembronic Membrane

14 14 14 Glucose- 1-C Glucose-2-C Glucose-6 -C DPM DPM DPM

Compound Source Days 1.1. mole OM N 1.1. mole (fM N. l!t' mole ()M N

Guanine RNA 2 5,600t 500 7 5,400tl,200 6 5,ooot 300 7

Cytosine RNA 2 12, BOOt 200 3 7' 200 t 200 3 11, 600t. 400 3 Uracil RNA 2 12' 400t.l' 300 3 7,200t 200 3 11,700t 500 3

Deoxyribose DNA 1 29 , 000!:2,700 9 32,200t4,500 7 36,200t3,400 9 Deoxyribose DNA 2 28,200!:: 600 7 31,400 l::2' 200 6 38,800t3,000 7

Adenine DNA 2 12, 700rl,500 7 10,20011,100 6 13' 500 tl, 300 7 Guanine DNA 2 10,900tl,300 7 lO,OOOt 800 6 10,400S:l, 200 7 62

embryo explants. Nearly all adenine samples were more heavily la­

beled than corresponding guanine samples.

The ratios of specific act1v1t1es of vea.rious fractions and com­

pounds 1n the embryo area to those In the membrane area are pre­

sented 1n Table X . Ratios were similar for a g1ven parameter , re­ 14 gardless of the position of c in the glucose substrate. The specific

activities of nearly all compounds , except those of the TCA extracts ,

were much greater in the embryo a r ea than in the membrane area.

The specific activities of the TCA extracts from the two areas were

nearly equal. The 6 specific activities of the embryo area wer e only

half of those of the membrane area, suggesting a difference in the

chemical composition between the two areas.

The ratios for ribose and deoxyribose samples increased f rom

24 to 48 hour explants, but underwent a slight decrease in the case of

RNA. The ratios of 6 speclfic activ1ties for RNA and DNA were near unity in the 48-hour explants, but less than unity in the 24-hour ex-

plants.

The specific activities in various fractions and nucleic acid hy­

drolysis products, arising from differently labeled glucose substrates, . 14 14 14 are compared as rat10s from G-1-C : G-6-C , and fr om G-2-C : 14 G-6-C , in Table XI. These ratios are not significantly different from unity in the TCA extracts, but appear less than unity in the etha­ nol: chloroform extract from the membrane area. The ratios from 14 14 G-2-C to G-6-C are less than unity in the protein fraction, and

greater them unity in RNA. Table X. Ratios of Specific Activities of-Various Fractions and Nucleic Acid Hydrolysis Products from Lab~led Glucose in Embryo:Membrane Areas

14 14 14 Fraction or Glucose-1-C Glucose-2-C Glucose-6-C Compound Source Days Ratio oM N Ratio oM N Ratio OM N

TCA Extract 2 1.07 t .06 8 0.92 -±: .06 6 0.93 t: .07 8 TCA Extracti> 2 0.50 ±- .03 8 0.43 :t .06 6 0.47 :t .04 8 CH CH 0H:CHC1 ' 3 2 3 Extract 2 3.84 t .06 8 4.34 ~ .87 6 3.14 -:- .41 8 Protein 2 1.07 -:. .11 7 1. 75 t .17 6 1. 81 t .08 7 RNA 1 2.04 t .06 8 2.13 !:: .08 7 2.08 ....- .06 7 RNA* 1 o. 77 t .04 8 0.84 ! .03 7 0.81 t .02 7 RNA 2 1. 73 -r .06 7 1. 86 -!; . 08 6 1.88 -±: .12 7 RNA* 2 1.05 = .07 7 1.05 t .02 6 1.04 !: .06 7 Ribose Adenosine 1 0.94 1 1.06 1 0.94 1 Ribose Guanosine 1 1.14 1 -- 1 0.90 1 Ribose Cytidine 1 1. 86 t: .97 2 1.35 ;t .54 2 1.40 t . 70 2 Ribose Uridine 1 1. 25 t . 23 2 1.07 ! .04 2 1.48 t .02 2 Ribose Adenosine 2 1.64 t .22 7 1.58 :t .16 6 1.54 + .11 6 Ribose Guanosine 2 1.65 t . 14 7 1.63 t .19 5 1.64 + .10 6 Ribose Cytidine 2 1. 87 -!:; .15 6 1. 79 t . 23 5 1. 79 t .14 7 Ribose Uridine 2 1. 73 !: .15 7 1.82 :!: .10 6 1.77 t .16 6

Adenine RNA 1 1.50 !: 1.00 2 0.43 -:. .15 2 0.94 1 ... + Guanine RNA 1 1.40 - . 90 2 0.47 ··- .20 2 Cytosine RNA 1 1. 76 !:: .48 2 2.45 .27 2 1. 72 +- .03 2 Uracil RNA 1 1. 25 t .03 2 1.05 =-!:; .45 2 1.43 :!: .24 2

• Table X. Ratios of Specific Activities of Various Fractions and Nucleic Acid Hydrolysis Products from Labeled Glucose in Embryo :Membrane Areas (Continued)

-­ - - -· -... ~ .. ··· 14 14 14 Fraction or Glucose-1-C I Glucose-2-C Glucose-6-C Compound Source Days Ratio N r ·­ Ratio N N oM ! OM Ratio QM - Adenine RNA 2 1. 48 ~ . 07 7 1. 48 ± . 16 6 1.54 t .06 7 Guanine RNA 2 1.89 -!:; . 12 7 1. 94 "!. .17 6 1. 93 -!;; . 16 7 Cytosine RNA 2 2 ~ 2 1 ± .12 3 1.53 -t . 03 3 1.47-!: .05 3 Uracil RNA 2 2.26 -!:; . 25 3 1.56 -.!: . 07 3 1. 54 -= . 07 3

DNA 2 1.53 1 .10 7 1.49 t: . 09 6 1. 29 -.!: . 16 6 DNA* 2 1. 04 -t .07 7 1.02 = . 07 6 0 . 89 t: ,11 6

Deoxyribose DNA 1 0.89 -:­ .03 8 0 . 97 t . 05 6 1. 00 "!: . 05 8 + Deoxyribose DNA 2 1.44 - .07 7 1. 45 ± . 04 6 1. 28 t. . 05 7 .... Adenine DNA 2 1.60 -= .11 7 1. 72 - . 22 6 1.32 ± .12 7 Guanine DNA 2 1.64 -= .10 7 1.81 = . 07 6 1. 79 ± .19 7

* Specific a ctivity was calculated as described in text. Table XI. Ra tios of Specific Activi ty of Various Fractions and Nucleic Acid Hydrolysis Products from Di fferently Lab e led Glucose-c14 Substrates. 14 14 14 14 Glucose-1-C . Glucose-6-C Glucose-2-C . Glucose-6-C Fraction or Embryo Membrane Embryo Membrane Compound Source Days Ratio N Ratio N Ratio C5M CJM CJM N Ratio ()M N --- TCA Extract 2 1.13 t . 06 8 0 . 97 t . 06 8 0 . 97 t .05 6 0 . 94 t . 08 6 CH CH 0H ; CHC1 3 2 3 Extract 2 0 , 99 t . 07 8 0 . 83 -= . 06 8 0.99 !: . 07 6 o. 77 ± . 0 7 6 Protein 2 0 . 95 t . 10 7 1.00 t . 09 7 0. 74 + .04 6 0 . 81 t .09 6 RNA 1 0 . 95 t . 05 8 0.94 t . 03 7 1.10 t . 03 8 1.06 t .04 7 RNA 2 0 . 97 t , 06 7 1.04 t . 05 7 1.17 t .06 6 1.11 t . 03 6

Ribose Adenosine 1 0 . 78 1 0.78 1 1.00 1 0.89 1 Ribose Guanosine 1 0 . 89 1 0.72 1 1.02 1 -- Ribose Cytodine 1 0 . 74 t .05 2 I 0.57 t . 02 2 0 . 97 +­ . 04 2 0. 95 +­ .07 2 Ribose Uridine 1 0 . 78 t . 05 2 I 0.94 t . 07 2 0.86 t .08 2 1. 23 t .28 2 Ribose Adenosine 2 0 . 88 t .03 7 0.95 t . 08 6 0 . 93 t . 05 6 0 . 97 + .05 6 Ribose Guanosine 2 0 . 86 t . 05 7 0.86 t . 05 7 0.96 t .03 6 0 . 98 t . 03 6 Ribose Cytidine 2 0.89 ± , 03 7 0.88 :!:" . 03 6 1.05 t .06 5 1.04 t .02 4 Ribose Uridine 2 0 . 85 ± . 04 6 0.90 t . 06 7 0. 95 + . 02 .5 0.99 t . 02 5

Adenine RNA 1 1.60 1 0.60 1 -- · 1.04 1 Guanine RNA 1 -- -- 1.00 1 -- Cytosine RNA 1 0.79 t . 08 2 0.85 t .12 2 0.87 t . 03 2 0.62 t .07 2 Uracil RNA 1 1.05 ± . 39 2 1.12 t .. 22 2 0. 79 t .03 2 1. 21 t .31 2 Adenine RNA 2 1.14 t .06 7 1. 20 t . 08 7 1.06 t .03 6 1. 20 ± .15 6 Guanine RNA 2 1.07 ± . 03 7 1.05 t .07 7 1.03 +­ .10 6 1.14 !" .31 6 Cytosine RNA 2 1. 70 ± . 15 3 1.11 t .02 3 0. 65 t- .02 3 0.62 t .01 3 Table XI . Ratios of Specific Activ ity of Various Fractions and Nucleic Acid Hydrolysis 14 Products from Differently Labeled Glucose-c Substrates. (Continued) 14 14 14 14 Glucose-l - c : Glucose -6-C Glucose-2 -c : Glucose-6-C - - - -- ~---·- Fraction or Embryo Membrane ----Embryo Membrane Compound Source Days Ratio GM N Ratio OM N Ratio GM N Ratio (JM N

Uracil RNA 2 1.56 t: . 16 3 1.08 t .12 3 0.63 t .03 3 0.62 t . 01 3 DNA 2 0.98 t .08 7 0 . 92 -t .13 7 1.13 + .04 6 0 . 99 +- .13 6 Deoxyribose DNA 1 0.73 t , 03 7 0 . 80 ± . 03 9 o. 95 ± . 02 7 0.92 t .OS 7 Deoxyribose DNA 2 0.81 t . 03 7 0, 73 -t . 05 7 0. 95 -;!;; . 03 6 0 . 83 t .04 6

Adenine DNA 2 1.17 ! .12 7 0.97 t . 10 7 1.07 ± .10 6 0 . 92 ! .13 6 Guanine DNA 2 1.19 ;; .20 7 1.09 t .07 7 1.00 t .05 6 1.08 t . 10 6 67 14 All ribose samples were l a beled more heavily from G-6 - C 14 than from G - 1- C , indicating some part icipation of the phosphoglu­ c onate pathway. The ratio of specific activities of ribose from G-l­ 14 c14 to G-6-c is greater in the 48 - hour than in the 24- hour explants, indicating a larger contribution of the non- ox idative reactions in the later embryo explants than in the e arlier ones . Ribose was almost 14 14 equally labeled by G - 2 - C and G - 6-C . No major differences in ribose specific activities, with respect to the base linkages of l;'ibo­ sides, are apparent.

The purine bases of RNA and DNA were more heavily labeled 14 14 14 from G - 1-C and G-2 - C than from G - 6 - C . The pyri midine bases 14 of RNA were much more heavily labeled from G - 1- C than from G-6­ 14 14 c14, and from G-6-c tha n from G-2 .,. c . 14 14 14 The 1ncorpora.t1on· · · o f c f rom R - 1- c , R - u - c , and f rom py­ ruvate - l - C14 into various fractions and nucleic acid hydrolysis pro­ ducts of the chi ck embryo explant are reported in Table XII. Compar­ 14 ison of these results w i th those for glucose - c in Table VI indicates 14 that c from none of these substrates is incorporated into the TCA extract, the ethanol: chloroform extract, or the protein fraction to the extent of glucose carbon. Ribose carbon is incorporated into RNA of the membrane area to a greater e x tent, and into RNA of the embryo area to a somewhat lesser extent than glucose carbon. I ncorporation 14 14 of c from pyruvate - l-c into all c hemi cal fractions except protein was minor. The distribution of pyruva te label among the various frac­ 14 tions is simi lar t o that for glucose. Pyruvate - l - C gave r i se to much 14 14 14 14 Table XII . Incorporation of c from Ribose-l-c , Ribose-u-c , and Pyruvate-l-c into Two-Day Chick Embryo Exp l ants. 14 Percent of Administered c Found in Various Fractions 14 14 14 Ribose-l-c Ribose-u-c Pvruvate-1-C Fraction Embryo Membrane Total Embryo Membrane Total Embryo Membr ane Total

TCA Extract 0.27 0.42 0,56 0.29 0. 55 0.84 0.17 0.37 0.54 CH CH 0H:CHC1 3 2 3 Extract 0.13 0.14 0.27 0.14 0.23 0.37 0.11 0.25 0.36 Protein 0.40 0.20 0.60 0.46 0. 20 0.66 0.36 0.30 0.66 RNA 0.40 0.45 0.85 0.43 0 . 37 0.80 0.11 0 . 06 0.17 DNA 0.09 0.06 0.15 0.10 0 . 06 0.16 0.02 0.01 0.03 Total 1. 29 1. 27 2.56 1.42 1.41 2.83 0. 77 0.99 1. 76 Original Homogenate 1.64 1.56 3.20 1.39 1.44 2.83 0 . 70 0.91 1. 61

C0 1.12 2.16 16.0 2 Ta bl· e XII • I ncorpora t ~on. o f cl4 f rom R~"b. ose .., 1 -cl4, R~" b ose-U-C 14, and Pyruvate-1-C 14 into Two-Day Chick Embryo Explants (Continued) Specific Activity 14 14 14 Ribose-l-c Ribose-u-c Pvruvate-1-C Fraction Embryo Membrane Embryo Membrane Embryo Membr ane

TCA Extract 390 530 420 620 260 640 TCA Extract* 462 1,539 501 1,560 309 1,645 CH cH 0H:CHC1 3 2 3 Extract 430 200 340 205 370 280 Protein 91 31 117 72 62 65 RNA 129 150 115 111 33 25 RNA"~> 146 309 130 237 37 52 Ribose 44,100 44,700 44,400 47,500 5,850 4,200 Adenine 8,500 7,430 5,000 4,850 5,130 4,050 Guanine 6,350 3, 780 3,020 1,960 3,930 2,570 C-2'- and 3 ' -P 24,900 23,900 24,200 24,900 8,070 6, 780 U-2 ' ­ and 3 I -P 26,200 28,200 27,800 26,200 8,430 11,000

DNA 257 158 168 195 40 42 DNA-I• 285 249 182 316 43 68 Deoxyribose 37,200 23,800 39,800 34,900 9,250 5,900 Adenine 20,800 18,900 -­ 9,850 6,600 4,600 Guanine 17,500 16' 500 13' 100 5,900 5,000 4,070

"~•The Specific Activity was calculated as described in the text. 70 14 more c o than did label from glucose. The yield of c 14o from 2 2 14 14 R-U -C was about twice that from R-l-C , but on an individual car­

bon basis, C-1 of ribose appears to have been decarboxylated to about

two and one-half times the extent as an average carbon from R - U - C 14 .

The specific activities of RNA labeled by ribose carbon are les s

than those of DNA, but the ribose moiety of RNA was more heavily

labeled by ribose carbon than the deoxyribose moiety of DNA. Pyr u ­ 14 vate -1-C gave rise to mor e label in deoxyribose than in ribose. In

this regard, pyruvate carbon resembled glucose carbon. Purines of

either RNA or DNA were much more heavily labeled by R-l-c14 than 14 by R-U -C . The purine bases of DNA were much more heavilylabeled by ribose carbon than were those of RNA. 14 As seen in Table XIII, pyruvate-1-C was a much poorer pre­ . 14 14 cursor of label in ribose or deoxynbose than R-1-C or R-U -C , but was nearly equal to ribose carbon with respect to specific activities in

RNA purines. Adenine was more heavily labeled than guanine by all three substrates. The pyr i di n e nucleotides were not hydrolyzed t o bases and ribose, but their s pecific activities arising from ~ibose -c14 indicate that the ribose moieties must be less radioactive than the ri­ bose which was associated with the purine bases. This was not true 14 for the pyruvate-1-C substrate, nor for the labeled glucose sub­ strates, as seen in Tables VIII and IX. 14 A study of incorporation of c from a two-carbon unit was made to ascertain whether a c2 + c3 m echanism for p entose formation might be operative to any extent in the chick embryo. The data from Table XIII. Ratios of Specific Activit ies of Various Fr actions and Nucleic Acid Hydrolysis 14 14 14 Products From Ribose-l-c , Rib o s e-u-c , and Pyruvate-l-c

Ratios in Embryo: Membrane Areas 14 14 14 Fraction Ribose-l-c Ribose-u-c Pyruvate-1-C

TCA Extract 0 . 74 0 . 68 0.41 TCA Extract'" 0 . 30 0 . 32 0.19 CH CH 0H:CHC1 Extract 2. 15 1. 66 1.32 3 2 3 Protein 2 . 94 1. 63 0 . 95 RNA 0.86 1.04 1.32 RNA''' 0 . 47 0 . 55 0 . 71 Ribose 0 . 99 0 . 93 1. 28 Adenine 1.14 1 ; 03 1. 27 Guanine 1. 68 1.54 1.53

C-2 I- and -3 I -P 1.04 0.97 1.19

1 1 U-2 - and -3 -P 0.93 1.06 0. 77 DNA 1.63 0.86 0 . 95 DNA* 1.14 0 . 58 0 . 63 Deoxyribose 1.56 1.14 1. 57 Adenine 1.10 -­ 1.18 Guanine 1.06 2 . 22 1. 23 Table XI II. Ratios of Specific Activiti es of Various Fr actions and Nucleic Acid Hydrolysis 14 14 14 Products· From . Rib ose - 1 -c , R'bL ·.ose · .-u -c , an d.. Pyruva •· · ·.t .~,., 1 ... c (cont1.nue · · d)

Ratios from Different Substrates 14 14 . 14 14 Ribose-l-c :Ribose-u-c R1.bose-U-C :Pyruvate- 1 - C Fraction Embryo Membrane Embryo Membrane

TCA Extract 0.93 0 . 85 1.62 0 . 97 CH cH 0H:CHC1 Extract 1.26 0.98 0 . 92 0.73 3 2 3 Protein 0.78 0,43 1.89 1.11 RNA 1.12 1.35 3 . 49 4 . 44 Ribose 0.99 0 , 94 7 . 59 11.31 Adenine 1. 70 1.53 0.97 1. 20 Guanine 2.10 1. 93 0. 77 0. 76

1 1 C-2 - and -3 -P 1.03 ·­ 0 . 96 3.00 3.67 U-2'- and -3 g, -P 0 . 94 1.08 3.30 2.38 DNA 1.53 0.81 4.20 4.64 Deoxyribose 0.93 0 . 68 4.30 5.92 Adenine -- 1.92 -- 2.14 Guanine 1.34 2.80 2.62 1.45

-....! N 73 14 14 ribose-c and pyruvate-l-C incorporation suggest that such a mech­ anism might be more important for the synthesis of deoxyribose than 14 14 14 for ribose . The incorporation of c from acetate-l-C and G-2-C into various fractions and nucleic acid hydrolysis products of one and two-day chick embryo explants are presented in Table XIV. The mem­ . 14 14 brane area 1ncorporated mor e C from acetate -1-C than from G - 2­ c14, and this greater incorporation was mainly due to the activity in the ethanol:chloroform extract. In contrast to the earlier experiments, 14 the specific activity of DNA arising from G-2-C was only slightly higher than that of RNA, and the purines of DNA were labeled less heavily than those of RNA. The deoxyribose moiety of DNA was much more heavily labeled than the ribose moie ty of RNA, in agreement with earlier experiments. 14 Comparisons of specific activities arising from acetate-l-C 14 and G -2-C are seen in Table XV. The ethanol:chloroform extracts . 14 14 were much more heaVlly labeled by acetate-1-C than by G-2-C

The TCA extracts of both embryo and membrane areas, and protein from the membrane, but not the embryo area, were more heavily la­ 14 beled by G-2-C .

RNA and DNA from the embryo area were labeled by acetate-l­ 14 c14 to less than one-half the extent as from G-2-C . Nucleic acids from the membrane area were from two-thirds to three-fourths as . 14 14 heaVlly labeled from acetate-1-C as from G-2- C . Most of the label 14 14 in RNA and DNA arising from acetate-l-C was due to the high c 14 content of the bases, while most of the label arising from G-2-C was 14 14 14 Ta bl e X.IV , I nc orporat~on. o f c f rom Ac etate­ 1 -C an d G1 ucose­2 -C into One and Two-Day Chick Embryo Exp lants DPM per Emb r yo 14 14 Substrate: Acetate-1-C Glucose-2-C Area: Embryo Membrane Embryo Membrane Days: 1 2 1 2 1 2 1 2 Fraction TCA Extract 940 1,870 3,180 3,620 1,860 2,230 5,060 4,750 CH cH 0H:CHC1 Extract 5,510 12,800 8,050 1.5,160 3,070 6,980 3, 720 5,800 3 2 3 Protein 1, 540 3,980 3,900 6,090 1,940 4,990 3,230 4,470 RNA 1,360 3,200 1,440 3,150 2,780 7,140 2,230 4,290 DNA 280 770 320 690 600 1,730 480 910 Sum 9,620 22,620 16,890 28' 710 10,250 24,070 14,720 20,220 Original Homogenate 9,550 22,250 17,300 29,350 8,920 25,880 14,810 23,020 14 14 14 Table XIV , Incorporation of C from Ac etate-1-C and Glucose-2-C into One and Two-Day Chick Embryo Explants (Continued) Specific Activi ty 14 14 Substrate: Acetate-1-C Glucose-2-C Area: Embryo Membrane Embryo Membrane Davs: 1 2 1 2 1 2 1 2 Fraction TCA Extract 352 308 551 316 603 536 717 460 CH CH 0H:CHC1 Extract 549 486 3 2 3 710 1, 185 305 345 324 403 Protein 118 148 117 158 147 201 85 107 RNA 77 92 53 98 164 200 85 121 Ribose 5,850 8,100 6,900 7,050 44,300 75,300 45,900 58,200 Adenine 5,540 7,560 3,510 6,750 10,500 16,500 5,810 10 ,400 Guanine 3,930 5,440 2,870 4,980 4,830 6,950 3,320 5,440 1 1 C-2 - and -3 -P 8,720 12,900 6,780 10,300 26,500 38,100 12,900 19,100 1 U-2 - and -3 I -P 10,400 18,800 10,000 21,400 29,200 54,400 12,000 24,000 DNA 79 89 94 113 177 212 136 151 Deoxyribose 11,000 16,800 10,100 10,200 83,500 175,000 65,500 -­ Adenine 5,130 5,130 2,430 3,380 5,260 11,140 2,840 -­ Guanine 4,680 4,680 2,420 2,870 4,540 5,590 2,570 -­ Table XV . Rat ios of Specific Activit ies of Various Fractions and Nucleic Acid Hydrolysis 14 14 Products from Acetate-l-c and Glucose-2-c Ratios in Embryo:Membrane Areas 14 14 Substrate: Acetate-1-C Glucose-2-C Da_ys: 1 2 1 2 Fraction TCA Extract 0.64 0.97 0.84 1.17 CH CH 0H:CHC1 Extract 0. 77 Oo41 0.94 0,61 3 2 3 Protein 1.01 0.94 1. 73 1. 88 RNA 1.45 0.94 1. 93 1. 65 Ribose 0.85 1.15 0 . 94 1. 29 Adenine 1.58 1.12 1.81 1.59 Guanine 1.37 1.09 1.45 1.28 1 1 C-2 - and -3 -P 1.29 1.25 2.05 2 . 00 1 1 U-2 - and -3 -P 1.04 0.88 2.43 2.27 DNA 0.84 o. 79 1.30 1.40 Deoxyribose 1.09 1.65 1. 27 -­ Adenine 2.11 1.5? 1.85 -­ Guanine 1. 93 1.63 1.77 -­ Table XV . Rat ios of Specific Activiti e s of Various Fractions and Nucleic Acid Hydrolysis 14 14 . Products from Acetat e - 1-C and G1ucose-2-C (ContLnued) . 14 14 RatLos from G1ucose-2-C :Acetate-1-C

Area : Embr yo Membrane Davs: 1 2 1 2 Fraction TCA Extract 1.71 1. 74 1.30 1.45 CH cH 0H:CHC1 Extrac t 0.56 0.50 0.46 0. 34 3 2 3 Protein 1. 25 1.36 0.72 0.68 RNA 2 . 13 2. 17 1. 60 1. 24 Ribose 7.57 9 . 30 6 . 65 8 . 26 Adenine 1. 90 2.18 1. 66 1.54 Guanine 1. 23 1. 28 1.16 1.09 c.,.2 ' ­ and -3 1 -P 3.04 2 . 95 1. 90 1.85 1 1 U-2 - and -3 -P 2.81 2.89 1. 20 1.12 DNA 2.24 2.38 1.45 1.34 Deoxyribose 7.59 10.42 6.49 -­ Adenine 1.03 2.17 1.17 -­ Guanine 0.97 1.19 1.06 -­ 78 due to the ribose moiety. The ribose of RNA was labeled by G-2-c14 14 from seven to ten times as heavily as from acetate-1-C . This ratio was nearly the same in the deoxyribose moiety of DNA. Purines of

RNA and DNA were more heavily labeled by G-2-c14 than by acetate­ l-C14. The differences are particularly large in the case of adenine. . 14 14 The ratio of specific activities ansmg from G-2-C to acetate - 1-C is considerably larger in adenine from RNA than from DNA in the one- day embryo explants, but the ratio is nearly the same in two-day ex- plants. 14 The distributions of c in ribose of RNA, arising from G-1, -2, 14 and -6-C , are reported in Table XVI. Ribose samples from many experiments were pooled prior to the degradation. Distinctions were made between ribose arising from the different positions of label in glucose, between 24 and 48-hour chick embryo explants, between ern­ bryo and membrane areas, and between purine and pyrimidine nucleo­ side sources. Workers have reported different labeling between purine-bound ribose and pyrimidine-bound ribose in the rat (135) and in Candida utilis (6). It was judged that there was not sufficient total radioactivity in any pooled sample to permit the degradation of dupli­ cate samples. 14 Most of the label from G-l-C appeared in C-1 of ribose, with the majority of the remainder in C -5. More randomization of label was found in ribose samples from two-day than from one-day embryo explants. In both one and two-day explants, slightly more label was found in C -1 of ribose, arising f rom G-l-C14, in the membrane area than in the embryo area. 14 Tab l e XVI . Distribution of c in Ribose of RNA* Hours Explanted: 24 48 Area of Blast oderm: Embryo Membrane Embrvo Membrane Base Linkage: Purine Pyrimidine Pur ine Pyrimidine Purine Pyrimidine Purine Pyrimidine Position of Label in Ribose from G-l-cl4: 1 86 88 94 91 65 62 74 77 2 4 6 2 3 4 9 14 7 3 1 1 1 1 4 1 Trace 2 4 Trace ' 1 Trace Trace 3 28t Trace 1 5 9 4 3 5 24 ? 22 13 Position of Label in Ribose from G-2-cl4: 1 50 51 43 48 16 19 22 11 2 38 37 43 46 68 67 61 78 3 1 Trace 1 1 1 1 1 4 } 12t 9 12 4 14 10 16 9 5 2 2 1 1 3 Trace 1

Position of Label in Ribose from G-6-cl4: l 1 4 1 7 3 5 9 8 2 Trace Trace Trace 1 3 4 9 7 3 1 1 Trace 2 3 10 5 4 4 1 1 1 4 5 2 1 15 5 97 94 98 86 86 79 76 66

*Data are reported as percents of the total specific activity of all carbons. tDetermined by difference when individual carbon samples were lost. 80 14 About one -half of the label from G-2-C appeared in ribose C -1 for one-day explants, and 10 to 20 percent in C-1 for two-day explants. 14 The remainder of the c appeared mostly in C-2, with significant amounts in C-4, and traces in C-3 and C-5. 14 Nearly all of the label from G-6-c appeared in C -5 of ribose 14 from one -day embryo explants. About three - fourths of the c in cor­ responding ribose samples from two-day explants appeared in C - 5, with the remainder distributed among the other carbons somewhat at random.

DISCUSSION

The positions of glucose carbon appearing in ribose formed by the oxidative and non-oxidative routes are shown by reactions 1 and 2, respectively, in Figure 8. Details of the reactions are given in the literature (8, 30, p. 431, 432, 438; 57, p. 236) . Complete molecular equilibration among ribose-5-phosphate, xylulose-5-phosphate, and ribulose-5-phosphate (and between dihydroxyacetone-1-phosphate and glyceraldehyde -3 -phosphate) is assumed for the frequencies of the three types of distribution found in ribose formed non-oxidatively.

Indications that complete equilibrium may not be attained have been published (45, 52, 82, 90). The relative specific activities of ribose 14 14 14 carbons arising from G-l-C , G-z-c , and G-6-C vary with the percent contributions by the two pathways as shown in Figure 9. The relative specific activity is the specific activity of the ribose carbon divided by that of the administered glucose. The values are altered 81 Figure 8 . Reactions of the Pentos e Cycle. Reaction 1: 1 c I 2 c 2 c I I 3 c Oxidative routjh. 3 c I -1 co I 4 c 2 4 c I I 5 c 5 c I I 6 c 6 c Hexose Pentose Reaction 2 : 1 c I 2 c l c 3 c 1 I c J I 6 3 c Non-oxidative 3 2 c + 2 3 c + 1 2 ~ I route I I 4 c ~ 4 c 4 t 3 c I I I I 5 c 5 c 5 c 2 c I I I I 6 c 6 c 6 c 1 c Hexose Pentose Pentose Pentose Reaction 3: 1 c l c 1 c I I I 2 c Recycling of 2 c 2 c I pentose cycle I I 6 3 c 2 1 c + 2 2 c + 2 3 c I I I 4 c 3 c 3 c 4 c I I I I' 5 c 4 c 4 c 5 c I I 5 c 5 c Pentose Hexose Hexose Triose Reaction 4! 5 c Carbohydrate skeletons are I 2 3 c 4 c labeled according to the original I I glucose carbon positions in reac ­ 4 c 3 c tions 1 and 2, and according to I I the pentose 5 c 3 c carbon positions in I reactions 3 and 4. 4 c I 5 c Triose Hexose 82

14 Figure 9. The Position of c in Ribose Derived From G-1, -2, or -6-c14 as a Function of the Percent Non-oxidative Pathway.

14 1.00 Glucose-l-c

0.75

0 . 50

0.25

0.00 Glucose- 2-c14 >. 1.00 .....~ c -5 .....> 1 ~ 0.75 (.) < .....(.) o.so 1+-4..... (.) Q) 0. tf') 0,25

Q) .....> o.oo ~ 111 .-1 14 Q) Glucose-6 - c p::: 1.00 ) c 1 - 5 0.75 o.so

o. 25 o.oo 0 25 50 75 100

Percent non-oxidative pathway 83

slightly when the substrate has been diluted by hexoses arising from

recycling by reaction 3 or reactions 3 and 4 of Figure 8. The positions

of ribose carbon appearing in the hexoses are indicated. 14 The distributions of c in the hexose pools altered by recycling from pentoses formed oxidatively were c alculated by Katz and Wood

(64, p. 2165-2177). Their calculations were based on the assumption of an isotopic and metabolic steady state. They considered the pentose cycle as a balanced equation involving the oxidative reaction 1 plus the recycling reaction 3, and did not consider the non-oxidative reaction

2 or reaction 4. Their results are expressed as the relative specific activities of the carbons of hexose pools obtained at various values of

"percent pentose cycle. 11 The dilution of the substrate hexose by hex­ oses arising from pentoses formed non-oxidatively gives rise to label­ ing patterns which are quite different from those calculated by Katz 14 and Wood. No alteration of the position of hexose c occurs when G­ l-C14 is diluted by recycling following oxidative pentose formation, 14 while c will appear in C-1, C-3, and C-6 following non-oxidative 14 14 pentose formation. The c from G-2-C will appear in C - 1, C-2, and C -3 of hexose by recycling following the oxidative reactions, while it will appear in C-2, C-3, and C-5 by recycling from pentoses formed 14 non-oxidatively. The position of c 14 from G -6-c will not be altered by recycling from pentoses formed by either route, unless reaction 4 14 of Figure 8 occurs. In that event, a small amount of c will appear in C-1 of the hexose pool. The differences in the labeling patterns of the hexose pools due to the different routes of pentose formation 84

indicate that the term "percent pentose cycle, 11 as used by Katz and

Wood, is not applicable when both routes are operatlve.

The extent of dilution of the hexose substrate by reformed hex­

oses is probably very small in a system such as the early chick em­

bryo, which is rapidly oxidizing glucose a nd synthesizing nucleic acids.

Synthesis of significant amounts of glycogen by embryos incubated less

than four days was not demonstrated (76), a lthough Rinaudo (112) accum­

ulated evidence that embryos older than six days, accumulating glyco­

gen rapidly, probably utilized t riose phosphates to a great extent for

glycogen synthesis. It is interesting to note that Hiatt discovered two

cases of similarity in ribose labeling patterns from ribose of acid sol­ 14 uble ribonucleotides and from RNA (54). He found that G-2-C gave

rise to almost identical labeling in ribose isolated from both sources

in rat urine and rat viscera. He also estimated that pentoses formed

~ in human carcinoma gave rise to pentoses of RNA with no further re­

cycling.

As seen in Figure 9, the relative specific activity of ribose aris­ 14 ing from G-6-C is independent of the percent non-oxidative pathway, 14 while that of ribose from G-lc is directly dependent. Interpreting the

percents ofpathways by the ratio of specific activities of ribose labeled by

14 1 G-l-C :G-O-C ~ the data onTable XI indicate a 75 to 80 percent non-oxida­

t i ve r oute for ribose ofone-day chickembryo explants, and 8 7 to 90 per­ 14 centfor two-day explants. The c distributions in ribose arising f rom 14 G-2-C vary considerablywith the percent non-oxidative route. As seen

in Figure 9, the relative specific activity in C -1 of ribose a rising from 85 14 G-2-C is directly proportional to the percent oxidattve route in

the case of no dilution by recycling reactions. Interpreting the per­ 14 cents of pathways by the percent of c in C -1 of ribose derived from 14 G-2-C , the degradation data m Table XVI indicate that the non-oxida­

tive route accounted for 43 to 51 percent of the ribose from RNA of one

day explants, and 78 to 89 pe:rcent for two-day explants. The two

methods of estimation provide evidence for a trend toward more non-

oxidative pathway participation as the embryo develops. Differences

between embryo and membrane areas, and between base sources, oc­

curred, but were generally within the experimental errors expected

for duplicate determinations.

The discrepancies between the estimations made from the rela­

tive specific activities of ribose arising from G-l-C14 and G-6-c14,

and the estimations made from the distribution of c 14 in ribose arising 14 from G-2-C , were small for the two-day explants. The discrepan­

cies w e re significantly larger for the one -day explants. In fact, some

workers have obtained results which are not explainable by the path­

ways outlined in Figure 8. Kit, Klein, and Graham observed that G-l­

c14 gave rise to more label in ribose of lymphatic tumor tissue than 14 G-6-C (68}. This effect could be reversed by keeping the coneentra­

tion of glucose very low. Barker, et. al., found that pyrimidine-bound 14 nbose was more heavily labeled by G-l-C than by G-6-c14inCandida

utilis (6}. The reverse was true for punne-bound ribose. When ade­

nine was added, the pyrimid1ne -bound ribose was less heavily labeled 14 14 by either G-l-C or G-6-c , and the ratio of labeling by G - l-C14 to 86 14 that by G-6-C was less than unity. A simllar effect was not observed in the case of purine-bound ribose. 14 14 The distribution of c in nbose derived from G-2-C was de­ termined in human carcinoma cells by Hiatt (53}. His data was very similar to that obtained in the present work wlth two-day chick embryo explants, and similar conclusions were drawn by Hiatt. Marks and 14 Feigelson studied the distribution of c in ribose arising from G- 2­ c14 in rat liver (90}. Their data is also similar to the data obtained for the two-day chick embryo explants, with the exception of C-.4 and

C-5 of ribose. No radioactivity was observed in these two positions of rat liver ribose. The workers concluded that ribose was formed by a combination of the oxidative and non-oxidative routes. The fact thatno label appeared in C -4 indicates that the dihydroxacetone-!-phosphate moiety resulting from glycolysis d1d not equilibrate with glyceralde ­ hyde -3-phosphate to serve as an acceptor for the transaldolase r eac ­ tion. In contrast to their results , Sokatch observed equal amounts of 14 c in C - 2 and C -4 of the ribose of Stre ptococcus faecalis, incubated 14 with G-2-C (124}. They could not demonstr ate transaldolase in this organism, and proposed a mechanism involving only transketolase. 14 Hiatt found nearly as much c in C-5 as in C-4 of the ribose of rat 14 14 liver RNA administered G-2-C (55}. Only 12 percent of the total c appeare d in C -1, and yet he concluded that the oxidative route accounted for 20 percent of the ribose formed. Bloom, also working with rat liver, found 23 percent of the total radioactivity in C - 1 of ribose aris­ 14 ing from G-2-C (17}. He concluded that equal participation of the two pathways of ribose formation occurred. 87

The works c1ted above indicate that all workers do not agree on the quantitative interpretation of r1bo s e labe ling, and that considerable l atitude must b e exercised in the quantitative evalua tion ofthe pathways involved. 14 The estimation from the c distr1bution data would be altered somewhat if the extent of dilution of substrate hexose by recycled hex­ 14 oses were known. This c an be approximated from the percent of C 14 not found in C -5 of ribose arising from G-6-C If the reactions in

Figure 8 represent the only reactions by which pentoses were formed 14 from hexoses, then label from G-6-C would appear only in C-5 of ribose, except when reactions 3 and 4 o c cur. Reaction 4 results in label in C -1 of hexose, wh1ch in turn will give rise to label in C -1 of ribose. The data indicate that this randomizing reaction could account for one to seven percent of the hexose precursor to ribose in one-day explants, and three to nine percent in two- day explants. However, the 14 majority of the label inC l-4 of ribose arising from G-6-c did not 14 appear in C -1, so other c randomizing reactions or other routes of 14 pentose synthesis must be considered. The amount of c appearing . 14 14 in positions other than those pred1cted from G-1-C , G-2-C , and 14 G-6-c varies from two to seven percent in the one-day explants. Un­ 14 14 predicted labeling from G-l- C and G-2-C in the two-day explants 14 varies from two toll percent, and from G-6-C from seven to 16 per­ cent. 14 c might contribute a significant amount of label to C - 3 and o 2 14 C-4 of hexose (90; 147, p. 475-489). Although c fixation by o 2 88

ribulose-5-phosphate was observed in the presence of 6-phosphoglu­

conic dehydrogenase isolated from yeast, giving 6-phosphogluconic 14 acid-l-C (58). the extent of this reaction is considered minor inmost

biological systems (30, p. 457}. The C0 fixation by pyruvate, how­ 2 ever, is fairly active in most tissues, and in one instance, actually

exceeded the extent of pyruvate entering the Kreb's cycle through con­ 14 version to acetyl coenzyme A ( 40}. Following the fixation of c o to 2 C-3 of pyruvate by the malic enzyme reaction ( 41, p . 146). the "C 4 shuttle, 11 involving interconversions of malate-;fumarate and mal ate_. 14 oxaloacetate, would lead to oxaloacetate-1, 4-C (41, 103, 139}. This

can b e decarboxylated, with the coupled phosphorylation by inosine tri­ 14 phosphate, to give phosphenol pyruvate -l-C (42, p. 512; 57, p. 232­

233}. Reversal of glycolytic reactions could yield fructose-!, 6-di­ 14 phosphate-3, 4-C , which could be dephosphorylated by fructose -!, 6­ diphosphate phosphatase to give fructose-6-phosphate-3, 4-c14. P e n­ toses formed non-oxidatively from this would be labeled in C-1, C-2, and C-3, while pentoses forn1ed oxidatively. foHowmg c onvers ion of fructose -6-phosphate to glucose-b -phosphate, would be labeled in C - 2 14 and C -3. Alternatively, glyceraldehyde-3-phosphate -3-c could ac ­ cept a c 2 unit from fructose - 6 - phosphate by means of the transaldo ­ lase reaction to form pentose labeled in C-3. Another c unit, formate, 1 might arise from any of the labeled carbons administered. Totter,

Volkin, and Carter found that 25 percent of the formate -c14 incorpora­ ted into pyrimidine nucleotides of rat liver RNA was in the ribose moiety (136). They found negligible activity in the purine-bound ribose. 89 14 Berstein observed that most of the c in RNA-ribose from chick, ad­ 14 ministered formate-C , appeared in C-3 and C-5 (12, p. 317-329).

Significant labeling also appeare d i n the other carbons of ribose. 14 14 Oxaloacetic acid-1,4- C would arise from G - 2 - C by the glyco­ lytic-Kreb' s cycle pathway (42, p. 515) . T his label could provide for labeling in pentose by the route described above. Oxaloacetic acid-2, 14 . 14 14 3-C would anse from G-1-C or G-6-C (42, p. 515). and would provide trioses labeled in C-2 and C-3 by the same route. These tri­ 14 oses could then give rise to pentose-4, 5-C by the transa1dolase reac­ 14 tion, or to hexose -1, 2, 5, 6 - C by the reversal of aldolase {42, p. 515).

Such a hexose could label all positions of ribose except C -3 by the non- oxidative route. This set of reactions would be more important for G­ 14 14. 1- C than for G-6-C 1f the pentose cycle reactions account for a large fraction of glucose utilization.

The ribose degradation data obtained admit all of the labeling possibilities discussed above. The extent of these possibilities may be 14 14 estimated from the incorporation of c from pyruvate - l-C . Label 14 from pyruvate -l-C could not appear in pentose as a result of direct 14 conversion to glyceraldehyde - 3 -phosphate-l-C by the reversal of glycolysis {64, p. 2165-2177). The "C shuttle, 11 following C0 fixation 4 2 14 by pyruvate, could be followed by conversion of oxaloacetate -1, 4-C 14 to phosphoenol pyruvate-1-C . Only one -half of the original specific activity in C -1 of the triose would result in the C0 originally fixed 2 were not labeled. Following conversion to glyceraldehyde-3-phosphate­ 14 1-C , pentose could be formed, by the transaldolase reaction, with a 90

maximum specific activity of one -half that of ribose labeled by R-U ­ 14 14 c . The observed specific activity in ribose labeled by R-U -C was 14 eight to 11 times that of ribose labeled by pyruvate-l-C . The mini­

mum ratio of pyruvate decarboxylated to pyruvate undergoing C0 2 fixation could be estimated from this data to be about 5:1. This ratio might also represent the amounts of ribose derived directly from car­ bohydrates and from the more devious routes, respectively. Consid­ 14 ering the fact that 16 percent of the c of added pyruvate appeared as 14 , and only 1. 6 percent was incorporated into the embryo, the in­ c o 2 14 corporation of c into ribose by the route described may not account for all of the observed activity. 14 14 The position of c in acetyl coenzyme A arising from G- 2-C traversing the glycolytic pathwaywould be identical to that from acetate­ 14 . 14 14 1-C . Approx1mately equal amounts of C from acetate-1-C and 14 G-2-C were incorporated into the embryo explants. Earlier experi­ 14 ments showed that about one and one-half times as muchC fromG - 2 14 14 -C appeared as than was incorporated into the embryo. About c o 2 14 14 one -half of the c incorporated into the embryo from acetate - l - C appearedinthe ethanol:chloroform extract, which consists primarily 14 of lipides {49, p. 12), whereas only one-third to one-fourth of the c 14 14 from G-2-C appeared in the ethanol:chloroform extract. If the c 14 in the ethanol:chloroform extract arising from acetate-l-C was mainly due to compounds formed from acetyl coenzyme A, then one might conclude that a much smaller fraction of glucose was converted to acetyl coenzyme A than that of acetate. On the other hand, acetyl 91 coenzyme A formed from acetate and from glucose might not be meta­ bolized by the same reactions. Evidence for a complex formed between acetyl coenzyme A and an enzyme following conversion of pyruvate to an acetaldehyde-thiamine phosphate complex, has been obtained by

Koike and Reed (73). This complex would arise from reactions involv­ ing decarboxylation of pyruvate, and probably would not arise to the same extent from acetate as from glucose. Acetate can be converted to acetyl coenzyme A by a single reaction involving energy from ATP

(42, p. 484). The intracellular locations of acetyl coenzyme A derived from acetate and from glucose by the different mechanisms might be different. Thus, one should not expect identical metabolic pathways for the c units derived from glucose and acetate. 2 14 If acetate -1-C were converted to acetyl coenzyme A and entered the Kreb' s cycle, the label would appear in C -5 of glutamate in the . 14 14 fust turn of the cycle (7 2). One -half of the C would appear as C 0 2 during the second turn of the cycle, one-half of the rema1nder during 14 the third turn, and so on. A significant fraction of oxaloacetate -1, 4-C 14 14 could be converted to phosphoenol pyruvate-1-C before C 0 could 2 appear, however. This label could be incorporated into pentose as 14 14 described above for pyruvate-l-c , yielding ribose -1, 2. 3 -c , with 14 more label inC-3 than the other positions. The ratio of c in ribose 14 14 derived from G-2-c to that from acetate-l-C is similar to the ratio . 14 14 from nbose - U -C and pyruvate-1-C Bernstein found that acetate­ 14 l-C labeled C-3 of RNA- ribose from chick organs to a much greater extent than any other carbons (12, p. 317-329). C -3 possessed 75 92 percent of the ribose label, C-2, 15 percent, C -1, seven percent, and

C-4, three percent. Qualitatively. stmilar r esults were obtained by

Bagatell, working with Escherichia cali, although the preponderance of label in C -3 was not quite as large as in the chick (3). 14 It would seem to require a longer t1me for c to be incorpora­ o 2 14 14 ted into ribose from acetate - l-c than from pyruvate -l-C by the 14 routes discussed. In fact, much of the c arising from any of the o 2 substrates might have been swept out of the system by the rapid flow of gas involved, or diluted by unlabeled C0 entering the chambers 2 containing the embryos.

Summarizing the discussion of acetate and pyruvate incorpora­ tion into ribose, one must conclude that at least 10 pe:rcent of the label in ribose may result from non-glycolytic and non-pentose cycle inter­ mediates. The immediate reactions leading to pentose from these intermediates may not necessarily be different from those given in 14 Figure 8. The similarities between incorporation of c from pyru­ 14 vate-l-C14 and acetate-l-C into ribose are probably due to the oxalo­ acetate intermediate formed by pyruvate + C0 on the one -hand, andby 2 conversion from acetate through the Kreb•s cycle on the other hand.

The biosynthesis of deoxy sugars was reviewed recentlybyGlaser

(43, p. 215-242). Although deoxyribose was not degraded in the pre­ sent studies, one might expectit to have labeling patterns similar to those of ribose. Several groups have obtained evidence that mammal­ ian and avian systems (3; 96, p. 651-653; 107; 108; 114, p. 439-454; 134} and bacterial systems (4, 14, 15, 48, 109, 110, ill) utillze the direct 93 reduction of ribose to deoxyr ibose. Reichard showed that in super na­ tant fractions of five-day chick embryo preparations deoxyguanosine ­

5' -phosphate and deoxycytidine-5' -phosphate are formed from the cor­ responding ribotides by an A TP-stimulated reduction (107, 108).

Reichard, et. al. (109, 110, 111) demonstrated that the reduction of pyr i ­ midine ribotides of Escher i chia coli. occurs at the diphosphate l evel, with an absolu te r e qui r e ment for reduced lipoic acid , A TP, and ma.g­ nesium (109). 14 Bagatell, et.~l., using acetate-l-C (3), and Bernstein and 14 14 Sweet, using G - 3, 4-C (1 4), found almost identical c distribution in ribose and deoxyribose isolated from nucleic acids of Escherichia c o li.

As discussed above, ribose from Escherichia coli. and chick, admin­ 14 ister ed acetate-l-C , had s imilar labeling patterns. Bernstein and

Sweet concluded f r om their wor k with Escherichia coli. that C - 1 of glucose was lost, and that ribose and deoxyribose came from a common precursor, or deoxyribose was der ived directly f r om ribose (14) . The y stated that "neither of the se expla n ations, howev e r , accounts for cer­ tain aspects of the dat a. "

Working with a purified Escherichia coli. enzyme, Racker dem­ onstrated that ribose-5- pho sphate was synthesized dir ectly from acet­ aldehyde and glyceraldehyde - 3 - phosphate (105, p. 347 -365) . Shreeve 14 and Grossman injected rats with glycine - l-and- 2 - C , followed by i s o ­ lation and degradation of deoxyribose from liver DNA (123). They c om­ 14 pared the c distribution to that found in the ribose of chick o r gan s by

Bernstein (12, p. 217-329). Shr eeve and Grossman concluded that rat 94

liver deoxyribose did not arise from glucose nor ribose, but from the

condensation of acetaldehyde and glyceraldehyde-3 -phosphate, as dem­

onstratcd in Escherichia coli. by Racker. Similar conclusions were

drawn by Boxer and Shank (19) in the case of liver and hepatoma cells,

and by Howells and Lindstrom (59) in Bac1llus cereus. Since Racker

worked with a purified enzyme system, and Reichard with a superna­

tant fraction of cells, it is possible that both mechanisms of deoxyri­

bose formation may occur in the same organism.

The ratios of specific activities arising from G-l-c14:G-6-c14 14 14 and from G-2-C :G-6-c , found in deoxyribose of DNA from the

chick embryo explants, were very similar to corresponding ratios 14 found in ribose of RNA. Specific activity ratios of C in the embryo 14 to c in the membrane areas were also very similar. These facts

indicate a synthesis of deoxyribose from ribose, or from precursors

common to both molecules. However, with all substrates except R-l­ 14 c14 and R-U -C , deoxyribose samples were more heavily labeled than

corresponding ribose samples. DNA samples from all labeled sub­

strates also had higher specific activities than corresponding RNA

samples, with the exception of the time course incorporation study. 14 The specific activities of DNA samples labeled by acetate - l - c and 14 G-2-C in the same experiment were only slightly higher than those of

RNA. In the latter two experiments, the DNA residues following the

RNA extraction were treated by much milder conditions than earlier experiments, as detailed in Methods.

Originally, it was hoped that deoxyribose, free of the 5-phosphate, could be recovered in good yield from the purine deoxynucleotides by 95 treatment of the DNA residue for one hour at 100°C in 1. 0 N HCl. Ac­ cording to Racke r , this treatment would hberate nearly all of the phos­ phate (105, p. 347-365). After this treatment, the apparent yields of

DNA, estimated by the diphenylam1ne reaction, were extremely low.

Attempts to recover free deoxyribose from paper chromatograms failed. Small amounts of diphenylam1ne-reacting material, corres­ ponding to the standard deoxyribose -5-phosphate spot on paper chrom­ atograms, were recovered. Good y1elds of adenine and guanine wer e obtained, however. The speciflc activities of such DNA extracts were considered anomolously high, due to the low yields of deoxyribose. In later experiments, DNA samples were treated with 0. 5 N HCl for 30 minutes at 90°C with the hope of recovering more of the deoxyribose-

5-phosphate. Yields of this compound, as well as apparent DNA, were better than before, and the apparent specific activit1es of DNA samples were lower. The specific actiVlties of deoxyribose -5-phosphate, fol­ lowing chromatography, were s1milar to those obtained earlier.

Even the latter treatment of DNA seemed inadvisable, in view o f a recent publication by L(\vtrup and Roos (83). These workers found that the destruction of deoxyribose with time exhibited an approximate first order rate curve. After 30 minutes at 100°C in 0. 5 N HClO4, only one -half of the original deoxyribose remained, as estimated by t.he diphenylamine reaction of Burton. Very similar results were ob­ tained with samples of DNA and deoxynucleotides. The problems of incomplete extraction of DNA with mild treatments, and the destruc ­ tion of deoxyribose with more severe treatments, were discussed by 96

L~vtrup and Roos at length. Using a microb1ological assay for com­ parison, they concluded that the Burton method is sufficient for extrac­ tion of nearly all the DNA, with very httle destructlon of deoxyribose.

Hutchinson, Downie , and Munroe reached the same conclusion after comparing the Schneider extraction method t.o the Schmidt and

Thannhauser method (60) . They showed that the apparent recovery of

DNA by extraction at 70°C. varied little with the concentration of

HClO , whereas at 90°C., a maximum of only 80 to 85 percent of the 4 DNA could be extracted, and this value dropped to about 50 percent when the concentration of HClO was increased from lN to 2N. 4 Hutchinson, Downie, and Munroe also determined that the recovery of

RNA was not lowered by excess1ve ac1d concentrations or temperatures, as estimated by the orcinol reaction. Yields of RNA extracted at 90°C. were slightly higher than at 70°C. Ribose is apparently quite stable to the severe hot acid treatments, while deoxyribose is not (120, p. 750).

The main degradation product of deoxyribose, under conditions more drastic than treatment in lN acid for 5 to 10 minutes at 60 °C. , is levu­ linic acid (120, p. 750).

In the light of these findings, it was considered inadvisable to make definite statements about comparisons of specific activities of

RNA and DNA samples. If some destruction of deoxyribose occurred while extracting DNA, then the apparent yield of DNA would be anomo ­ lously low, while the specific activity would be correspondingly too high. Indeed, the milder treatments in the time course experiment indicated that the specific activities of DNA samples were less than 97

those of RNA in the embryo area, and only slightly higher than RNA in

the membrane area. Specific activities of DNA in the acetate-l-C14

incorporation experiment were equal to or only shghtly higher than

those of RNA, while the calculated 6specific activities of DNA were

lower than those of RNA. Comparisons between specific activities of

DNA samples arising from one substrate to those of another, in the

same experiment, should s till be valid.

Comparisons of specific activities of ribose and deoxyribose

should be valid if no radioactive impurities were included in the eluants

of the paper chromatograms. This qualification must rest until it can

be determined whether the destruction product or products of deoxyri­

bose would appear at the same position as the deoxyribose - 5-phosphate

spot. Eluants of the paper chromatograms of the sugars, as well as

the aqueous effluents from the charcoal columns preceeding the paper

chromatography of the sugars, always gave a negligible test for pur ine

and pyrimidine compounds. 14 Since sampl es of d e oxyribose were labeled by all glucos e - C

substrates to a greater degr ee than corresponding ribose sampl es, one must tentatively conclude that the synthetic pathways of deoxyribose may differ from those of ribose. The ratios of specific activities of 14 ribose to deoxyribose were much greater than unity when R-l-C or 14 R-U -c were used. These ratios were much higher in the membrane than in the embryo area of the explants. These r esults point toward a contribution of synthetic pathways for deoxyribose different than the direct reduction of ribose, and contributions might be more pronounced 98 14 in the membrane than the embryo area. Pyruvate-1-C and acetate­ 14 14 l-C , similar to glucose-C , labeled deoxyribose more heavily than ribose. These results would be expected if the acetaldehyde + glycer­ aldehyde-3-phosphate mechamsm discovered by Racker contributed significantly to the synthesis of deoxyribose. Whereas the ratios of 14 specific activity of ribose:deoxyribose de rived from G-l-C and G-6­ 14 c were slightly greater in the embryo than in the membrane area, 14 the ratios were similar when G- 2-C was used, and were greater in 14 the membrane area when pyruvate-1-C was used. The ratio was much greater in the membrane than the embryo of one -day explants, 14 but much less in the two-day explants, when acetate-1-C was used.

These comparisons indicate that the contributions by different synthetic schemes must be different in the two areas.

Certain aspects of the labeling of the purines and pyrimidines isolated from the chick embryo explants appear to agree with the ac­ cepted synthetic routes for these compounds (42, p. 877 -906; 106, p.

263 -294). Pyrimidine positions 1 and 2 are derived from the nitrogen and carbon of carbamyl phosphate, respectively, and positions 3, 4, 5, and 6 are derived from the nitrogen, C-2, C-3, and C-4 of aspartate, 14 respectively (106, p. 263 -294}. Oxaloacetate -1, 4-C would be derived 14 14 . from G-2-C , and oxaloacetate-2, 3 -C would be denved from G-1­ 14 14 . C or G-6-C , by the glycolyhc route and one turn of the Kreb's cycle. Corresponding labels would appear in aspartate, following the transamination reaction. Since C-1 of aspartate is lost upon incorpor­ ation into the pyrimidine, the above route would label the pyrimidine 99 14 14 14 twice as heavily from G- 1-C or G-6-C as from G- 2- C . 14 If c , derived in equal amounts from all glucose substrates, was o 2 incorporated into carbamyl phosphate, the resultant pyrimidine would 14 be labeled between one and two times as heavily from G-l-C or G-6­ c14 as from G-2-C14.

Pyrimidines from the embryo area of two-day explants were 1 14 14 labeled by G-l-c \G-6-c : G-2-C in th e approximate ratios of 2. 5:

L 6:1. 0 , while corresponding ratios from the membrane area were about L 8:1. 6:1. 0. The observed ratlos suggest that the above scheme may be correct, with the exceptions of the higher specific activity in 14 14 pyrimidines labeled by G-l- c than by G-6-C , and the ratio of pyri­ 14 14 midine label from G-l-c :G-2-C greater than two. The ratio of 14 14 pyrimid1ne specific activity from G-l-C :G-6-c could be greater than unity if the carbamyl phosphate intermediate were labeled to a 14 14 14 larger extent from c o arising from G - 1-C than from G-6-c as 2 a result of the phosphogluconate pathway. The magnitude of the dif­ 14 ferences in pyrimidine labeling from G-l-C and G-6-c14 in the e m ­ 14 bryo area was considerably larger than the differences in c o 2 yields from the two substrates, suggesting that the phosphogluconate route could be more active in the embryo area thaninthe membrane a r ea

This, however, was not born out in the ribose degradation data dis ­ cussed above. 14 14 When oxaloacetate -1, 4-C is labeled by acetate-l-C , arising 14 from G-2-C via the glycolytic-Kreb 1 s cycle pathway, the specific activity in either C-1 or C-4 is one-half that of C-1 of acetate, due to 100

the randomization occurring when succinate 1s formed. This fact sug­

gests that label in carbamyl phosphate may contribute relatively more

label to the pyrimidine than the asparate intermediate. As discussed

earlier, however, the unlabeled C0 introduced into the system by the 2 14 aeration would tend to dilute the specific activ1ty of c o arising from 2 glucose oxidation. During the second turn of the Kreb's cycle, all of 14 14 the c incorporated into oxaloa.cetate from G- 2-C in the first turn 14 would be lost, whereas the label from G-1 or -6-C would be random­

ized among all four carbons, but none lost. This effect would be dim­

inished, as the acetate condensing with the labeled oxaloacetate formed

during the first turn of the Kreb's cycle would also be labeled. How­

ever, c onsiderations of repeated turns of the Kreb's cycle do permit 14 the possibility of oxaloacetate labeled by G-1 or -6 -c to have more 14 than twice the specific activity as that from G-2-C .

W1th the exception of the uracil isolated from the membrane area,

pyrmidines from one -day explants were also more heavily labeled by 14 14 G-1 or -6-C than by G-2-C . Differences in specific activities of 14 pyrimidines from one-day embryo explants, due to the different c

substrates, are less than in the two- day explants. This may be due to

a greater contribution of label by the carbamyl phosphate intermediate than the aspartate intermediate. As noted in Figure 7, the peak in 14 recovery from explants occurs at about 24 hours. However, c o 2 only two determinations are represented by the data for one -day ex- plants, and the values for the standard errors of the mean were large,

so it is difficult to conclude anything from the one-day explant experi­

ments for pyrimidine labeling with certainty. 101

Purine biosynthesis involves smaller compounds than pynrni­ dine biosynthesis. Three of the five carbon atoms arise from c units, 1 and two from glycine (42, p . 890) . The specific activities of RNA purines isolated from two- day embryos showed httle differences de­ 14 pending on the c -labeled glucose substrate used. Again, the results from one-day explants represent only one or two determinations, and the standard errors of the mean were quite large. From the two-day embryo explants, RNA purines were slightly mor e heavily labeled by 14 14 14 14 . G-1-C than by G-2-C , and by G-2-C than G-6-C . Th1s was also true for DNA purines. These results could occur if more C - 1 14 14 14 units from G-l-C and G-2 - C were utilized than from G-6-c .

However, if the pentose cycle were active, one would expect that C-2 14 14 units from G-6-c and G-2-C would be incorporated to a greater 14 extent than from G-l-c , giving the reve rse results. The forme r ef­ feet is supported by the incorporation into purines of both RNA and 14 14 DNA from R-l-C . This incorporation exceeded that from R-U -c by a considerabl e margin. Whereas trioses resulting from the pentose 14 14 . cycle would be unlabeled from R -1-C , C 0 derived from recychng 2 of the oxidative pentose cycle would account for the majority of label 14 14 fixed in purines from R-l-C Pyruvate-l-C gave r ise to much 14 more c than any other substrate investigated, and the RNA purines o 2 labeled from pyruvate were also more heavily labeled than those f rom 14 R-U -C . The DNA purines, however, were more heavily labeled by 14 14 R - U -c than by pyruvate-l-C , suggesting that DNA purines may arise by different routes than RNA purines. This possibility is also 102 suggested by the fact that the ratio of specif1c activities arising from 14 14 G-2-C : acetate-l-C was considerably larger in ademne from RNA than from DNA of one-day explants.

The possibility of different synthetic pathways for purmes of

RNA and DNA is interesting. The react10ns leading to purines begin with the formation of 5-phosphoribosyl-1-amine {42, p. 890). This nitrogen-glycoside linkage is not broken during the entire buildup of the purine ring, which results in a purine-5'-ribotide. However, if deoxyribose-5-phosphate can be synthesized by a c2 + c3 mechanism, perhaps the enzymatic reactions involved in the synthesis of the purine ring of the deoxyribotide are different from those for the ribotide.

The calculations of ~ specific activity for RNA and DNA were performed in order to gain a better comparison between embryo and membrane areas. The ~specific activities of the purines of RNA and

DNA were not calculated since the calculahons would involve unknown factors. The ratios of~ specific activity:original specific activity were not as large for DNA as for RNA because a lesser fold increase in RNA occurs during the explant period than that of DNA. The same trend would occur in the case of the purines and sugars of the parent nucleic acids, and would lessen the differences in specific activity be­ tween hydrolysis products of RNA and DNA.

Another possible explanation for the differences could involve different pools of nucleotides or polynucleotides. Henderson obtained evidence of intracellular compartmentation of purine nucleotides in

Ehrlich ascites tumor cells (51). He observed that ethidium bromide 103 inhibited the incorporation of preformed adenine, guanine, and hypox­ anthine into nucleic acids, but did not inhibit the formation and incor­ poration of these purines syntheslZed de ~· He concluded that the pool of purine nucleotides made from preformed purines was separated in some way from the pool of purines synthesized de ~· The works of Totter, Volkin, and Carter (135) and by Barker (6), cited above in­ dicate that purine-bound r i bose can be synthesized by different pa th­ 14 ways than pyrimidine -bound r ibose. In the acetate -l-C incorporation study, it was observed that the specific activities of RNA purines in­ creased from one -day to two-day explants, while those of DNA did not

The specific actlvities of ribose and deoxyribose from G-1, - 2, o r - 6­ c14 showed simllar trends. Specific activ1ties of deoxyribose sample s from the membrane area showed no apparent increase from one-day to two-day explants, while those of nbose underwent nearly a two ­ fold increase. In the embryo area, the specific activities of deoxyr i ­ bose samples increased about l. 5 fold from one to two-day explants , while the specific activities of cor responding r ib ose samples increased from two to three - fold.

The unincubated hen•s egg contains 310 llg. of RNA and only 2 10

~· of DNA (37, p. 46-57) . If these nucleic acids, present in the med ­ ium, can enter the embryo explant as large polymers, at about equal rates, then the ratio of the !J. specific activities of RNA:DNA would be much less than the ratios of observed specific activities. Another con­ sideration would be the content of acid soluble nucleotides of the uninc­ ubated egg. Emanuelson found considerable amounts of ribonucleotides 104

in the unincubated egg {37, p. 46-57) . Adenosme, guanosine, and ura­

cil ribonucleotides were found in the molar proportions of 9:26:65,

respectively. Negligible amounts of cytidine ribonucleot1des and deoxy­

ribonucleotides were found. If the ribonucleot1des could be incorpora­

ted into the embryo RNA from the substrate, but not converted to de­

oxyribonucleotides of DNA at the same rate as the rate of formation of

deoxyribonucleotides de novo from labeled precursors, this effect

could also account for higher speclfic activities in DNA samples a r ising 14 from glucose-C . Although Emanuelsson found more pyrimidine nu­

cleotides than purine nucleotides in the umncubated egg, the chick em­

bryo explant RNA pyrimidines, as well as pyrimidine-bound ribose, were more radioactive than corresponding purines and punne -bound

ribose. The reverse would be expected if unlabeled purine and pyrimi­ dine nucleotides diluted the pools of nucleotides synthesized de novo in proportion to their relative amounts. Since adenine samples were more heavily labeled by glucose carbon than corresponding guanine samples, the dilution of r a dioactive purine nucleotides by unlabel e d nucleotides was somewhat p r oportional to the relative amounts of the purine nucleotides found in the unincubated egg.

Since Reichard's work with supernatant fractions of five-day ern­ bryos indicates that this is not hkely, one should try to establish whe­ ther large polymers of RNA and DNA can be incorporated into chick embryo explants without initial breakdown to nucleotides. However, the indications that deoxyribose may be synthesized independently of ribose do present intriguing possibilities of different schemes for DNA 105 and RNA purine synthesis. Henderson's work, indicating separate pools of preformed purines and punnes synthesized de novo, might apply to purines of chick embryo DNA which might have to be formed de~ to a greater extent thc..n those of RNA. An approach to this question should involve the chem1cally defined medium designed by

Klein, McConnell, and Buckingham for the growth of chick embryo explants ( 71).

Certain differences between the utilization of labeled carbon by the embryo and the membrane areas were seen in the explant experi­ 14 14 ments. The rates of incorporation of c from glucose-6-C intothe embryo area were different than in the membrane area, as seen in

Figure 5. The r ate of incorporation into the e mbryo area continued to increase with time, while that of the membrane area decreased with time. The decrease of incorporation rate of the membrane area was seen to b e due to the plateau in the TCA extract, which appeared at about 12 hours after explanting. The initially r apid increase in total activity of the TCA extract of the membrane, compared to the slow continuous rise in the embryo, indicates that the membrane in­ corporated the labeled glucose to a much greater extent, initially, than the embryo. In fact, the TCA extract of the membrane after 6 hours represented over half of the total activity of the membrane, while protein of the embryo area at this time was more radioactive than the TCA extract. These facts md1cate an initial incorporation of glucose primarily into the m embrane, followed by transfer to the em­ bryo, where protein and nucleic acids are being synthesized at a 106 greater rate than in the membrane , The larger surface area of the membrane for absorption of substrate, rather than a more pronounced active transport m e chanism, could account for this effect. The pla­ 14 teau in the membrane TCA extract c after 12 hours could be the re­ sult of the attainment of an isotopic steady state condition, This con­ cept could not be strict ly valid in the case of the chick embryo explant; 14 since the rate of c o2 production decreased after 24 hours , while the 14 rate of incorporation of glucose -C did not.

The b. specific activities provide a better comparison between the embryo and the membrane than the original specific activities .

The specific activities of RNA and DNA were greater in the embryo than the membrane, while their b. specific activities were nearly equaL

The specific activities of both ribose and deoxyribose were sli~htly higher in the membrane than the embryo of one -day explants, but were higher i n the embryo of two-day explants . The fl.specific activities of the sugars would be much higher in the membrane than the embryo of one -day explants, based on the differences in original specific activ­ ities and specific activities of RNA and DNA. Since this is not true for purines and pyrimidines, perhaps the membrane area utilizes the synthesis of these bases from glucose carbon to a greater extent, and incorporation of preformed bases to a lesser extent, than the embryo. 14 Differences in the specific activities of pyrimidines from G-l-C :G­

6-c14 i n the embryo and the membrane areas actually point to differ­ ences in the specific activities of the carbamyl phosphate and the as­ partate intermediates of the two areas . 107 14 Distinct differences in the incorporat10n of glucose -C , ribose­ 14 14 14 c , pyruvate -l-C , and acetate -1 -C between the embryo and mem­ brane areas were noted, Ratios oft. spec1flc activ1ties of RNA in the embryo:membrane areas were near unity for labeled glucose substrates, 14 but much less than unity for labeled ribo ~<.> and pyruvate-1-C sub­ strates. The ratios of speciiic act1v1tles in embryo:membrane areas for protein were much greater than unity for labeled glucose and ri­ 14 bose substrates, but slightly less than unity for pyruvate-l-C and 14 14 acetate-l-C . The greater utihzatlon of acetate-1-C for prote1n synthesis in the membrane than the embryo might be due to the higher proportion of hemoglobin to total protein of the membrane than the ern­ bryo. The heme portion is denved entlrely from acetate and glycine

(47, p. 865). Ratios of specific activity in embryo:membrane TCA extracts were near unity for labeled glucose substrates, less than un­ 14 ity for ribose subst.rates and acetate -l-C from two-day explants, and 14 much less than unity for pyruvate-l-C of two-day explants and ace­ 14 tate-1-C of one-day expl ants .

The most striking differences in specific activities of the embr yo and membrane areas were noted in the ethanol:chloroform extract. Ra­ tios of three to four were obtained from labeled glucose substrates, 14 14 14 2. 2 from R-1-C , 1. 7 from R-U -C , 1. 3 from pyruvate-1-C , and 14 0. 4 to 0. 8 from acetate-1-C . Major constituents of the lipides ex­ tracted by ethanol and chloroform from the embryo would be brain sphingolipides (49, p. 134) and inositides (49, p. 106). Hanahan con­ siders the cerebrosides and ganglios1des as members of the 108 sphingolipide group (49, p. 134). The galactose moiety of cerebro­ sides (and the galactose, glucose, and hexosamine moieties of gang­ liosides) and the inositol moiety of inositides may be derived from glucose without breaking the carbon chain. As only traces of cere­ brosides were found in the unincubated egg ( 115, p. 27). the utiliza~ tion of glucose carbon for their synthesis would be a more important feature of the brain than of other tissues. The glycerol and fatty acid moieties of glycerides may be derived from smaller units.

They could account for the similarity in specific activities of lipides 14 from the embryo and membrane areas derived from pyruvate-l-C , 14 or the greater specific activity of lipides derived from acetate··l-C in the membrane than the embryo. Both long chain fatty acids and sterioids may be synthesized entirely from acetate (42, p. 627), and may account for a larger percentage of the compounds extracted by ethanol and chloroform from the membrane than the embryo.

SUMMARY

(1) RNA ribose of the chick embryo explant was found to arise 14 from c -labeled glucose primarily by the non-oxidative pathway. 14 14 Calculations based on the incorporation of c from G-2-C into ribose indicated 43 to 51 percent of the ribose from one-day ex­ plants and 78 to 89 percent for the two-day explants was formed non­ 14 oxidatively. Comparisons of C incorporation into ribose from 14 14 G-1-C and G-6-C indicated that the non-oxidative pathway accounted for 75 to 80 percent and 87 to 90 percent of the ribose of RNA from one and two-day embryo explants, respectively. 109

The discrepancies m the two methods of estimation of pathwaypartici­ pation were discussed. The contnbutJOn of non-glycolytlc and non- pentose cycle intermediates to the ultimate formahon of nbose carbon 14 was estimated from experiments utihzing pyruvate-l-C and acetate ­ 14 1-C to be about ten percent of the total nbose carbon. Label found in positions of ribose not predicted by theoretical cons1derat10ns sup­ ported this estimate. Little d ifferences in ribose labeling due to the embryo or the membrane area source of RNA, or to the purine or the pyrimidme base linkage source, were found.

(2) DNA deoxyribose of the ch1ck embryo explant might have arisen by reactions other than the direct conversion from nbose by reduction. Specific activities of deoxyribose samples derived from 14 14 . all substrates tested except R-1-C and R-U -C were h1gher than corresponding samples of ribose .

(3) The labeling of RNA and DNA purines and RNA pyrimidines 14 of the explant by different c substrates was investigated. A signif­ 14 icant contribution to base label by c o fixation was indicated. Dif­ 2 14 ferences in specific activities of pyrimidines labeled by G-1-C and 14 G-6-c indicated contributions by the phosphogluconate pathway in the initial utilization of glucose. Evidence was obtained to support the concept of separate pools of DNA precursors and RNA precursors.

Contr1but1ons to embryo explant DNA molecules by unlabeled precur­ sors appeared much more important in the second than the first day of explantation. 110

(4) Differences between the embryo and the membrane areas 14 with respect to the u t ilization of c - 1a b e led glucose, rib~se , pyruvate a nd aceta t e w ere found. The most pronounced differences occurred in 14 the incorporation of acetate -1-C into the ethanol: chloroform soluble materials of the two areas. The greater incorporation of a.cetate-l­ 14 c14 into the membrane a. rea lipides , and of glucose -c into the em­ bryo area. lipides, were interpreted as an indication of different types of lipide material in the two areas . The greqter incorporation of 14 14 acetate -1-c into the membrane protein, and of glucose -c into the embryo protein, was interpreted to be due i n part to the larger portion of the heme protein in the membrane than the embryo. The glucose -6­ c 14 incorporation rate studies indicated that TCA extrac;t of the mem­ brane area absorbs glucose at a far greater rate than the TCA extract of the embryo shortly after explantation. This fact, coupled with the fact that the i n i t ial l a beling of protein and nucleic acid was greater in the e mbryo, i ndicated that the embryo derives more of its nutrients by transfer from the membrane than by direct absorption from the substratum.

(5) The attainment of a proximate isotopic: steady state after 12 hours of explantation of the chick embryo was observed. Time course 14 14 studies of c o2 evolution from c -labeled gll,lcose indicated that a true isotopic steady state did not attain. The interval recoveries of 14 c o from the chick embryo explants administered G-1, -2, and -6 ­ 2 c14 indicated a small contribution to glucose utilization by the phospho­ 14 gluconate pathway. More interesting was the drop in c o recovery 2 111 following 24 hours of explanting. Since cellular constituents continued to increase in radioactivity, the drop indicated a change at about 24 hours to a less oxidative utilization of glucose by the explant, This was supported by the ribose labeling patterns . 112

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sphingolipide group (49, p. 134). The galactose moiety of cer~="bro··

sides (and the galactose, glucose, and hexosamine moieties of gang··

liosides) and the inositol moiety of inositides may be derived from

glucose without breaking the carbon chain. As only traces of cere~"

brosides were found in the unincubated egg ( 115, p. 27), the uLiizd.·~

tion of glucose carbon for their synthesis would be a morr:o irnportant

feature of the brain than of other tissues. The glycerol and fatty

acid moieties of glycerides may be derived from smaller units.

They could account for the similarity in specific activities of lipidts

from the embryo and membrane areas derived from pyruva.tt';-·l~C 14 , 14 or the greater specific activity of lipides derived from acetate .. 1.. c

in the membrane than the embryo. Both long chain fatty acids and

ste rioids may be synthesized entirely from acetate ( 42, p. 627), and

may account for a larger percentage of the compounds extracted by

ethanol and chloroform from the membrane than the embryo.

SUMMARY

(1) RNA ribose of the chick embryo explant was found to arise 14 from C -labeled glucose primarily by the non-oxidative pathway 14 14 C a l cul a t 1ons. b ase d on ·the 1ncorpora. t"1on o f c from G-2-C 1"nto

ribose indicated 43 to 51 percent of the ribose from one-da.y ex·­

plants and 7 8 to 89 percent for the two -day explants was formed non­ 14 oxidatively. Comparisons of C incorporation into ribose from ,14 14 G-1-C and G-6 -C indicated that the non-oxidative pathway accounted for 75 to 80 percent and 87 to 90 percent of the ribose of RNA from one and two··day embryo explants, respectively. 109

The discrepancies In the two methods of estimat10n of pathway partici­ pation were discussed. The contnbutron of non-glycolytrc. and non- c)entos c :. yclc:: rnl~_; rmedia te s to the ultrmate formatron of n base carbon 14 was estlmated from expe rimt-nts utlhzing pyruvate -1-C and acetate­ 14 1-C to be about ten perc cnt of the total nbose carbon. Label found in posrtlons of ribose not predicted by theoretical consrderatwns sup­ ported this estimate. Little drfferences in ribose labeling due to the embryo or the membrane arect source of RNA, or to the purine or the pyrimidine base linkage source, were found.

(2} DNA deoxyribose of the c.hrck embryo explant might have arisen by reactions other than the direct conversron from ribose by reduction. Specific actlvrtles of deoxynbose samples denved from 14 14 , all substrates tested except R-1-C and R-U-C were hrgher than corresponding samples of ribose.

( 3} The labeling of RNA and DNA purines and RNA pyrimidines 14 of the explant by different c substrates was investigated. A srgnif­ 14 icant contributwn to base label by c o fixation was indicated. Dif­ 2 14 ierences in specific activities of pyrimidines labeled by G-1-C and 14 G-6 -C indica ted contributwns by the phosphogluconate pathway in the init1al utilization of glucose. Evidence was obtained to support the concept of separate pools of DNA precursors and RNA precursors.

Cun• -· · jtrons to embryo explant DNA molecules by unlabeled precur­ sors appeared rnuch more important rn the second than the first day of e xplantation. 110

( 4) Diffe renee s between the embryo and the membrane a rea s 14 w1t. h respect to t1e1 uti"1 Ization ot- L· -la b cled glucose, ribose pyruvc:tte

cl.nd acel.ate were found. The rnust pronounced differences occurred In 14 the incorporatiOn of acetate -1- C Into the ethanol: chloroform S olub}e

mate rials of the two a rea s The greater Incorporation of acetate-1­ 14 14 c into the membrane d red lipides and of glucose -C into the em­

bryo area lipides, were interpreted as an indication of different types

of lipide material in the two areas. The greater incorporation of 14 14 acetate-l-C into the membrane protein. and of glucose-C into the

embryo protein, was interpreted to be due In part to the larger portion

of the heme protein in the membrane than the embryo. The glucose -6­

C14 incorporation rate studies Indu.;:ded that TCA extract of the mem­

brane area absorbs glucose at a far greater rate than the TCA extract

of the embryo shortly after explantation. This fact, coupled with the

fact C1at the initial labeling of protein and nucleic acid was greater In

the c:-nbryo Indicated the:tt the embryo denves more of its nutnents

by transfer from the membrane than by direct absorption from the

substratum.

(5) The attainment of a proximate isotopic steady state after 12

hours of explantation of the chick embryo was observed. Time course 14 14 studies of c o2 evolution from c -labeled glucose indicated that a tn.tc 1:-;utopic steady state did not attain. The interval recoveries of 14 C o.>c.- ±·ro1n the c·h1'ck em b ryo exp l ants a d mlnistere · · d G -,l -2 , an d -­6 14 c Indicated a small contributiOn to glucose utilization by the phospho­ 14 gluconate pathway. More interesting was the drop In c o recovery 2 11 l following 24 hours of e xplC!nt1ng. S1nce cell uL:, r constituents contlnued to increC!se 1n radioactJVJty tht drop Jncbcated a. change at about 24 hours to a less uxiddtiVt' ut1hzd:on of glucose by the explant This was supported by the ribose ;._,bellng patterns. 112

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