ISOLATION, IDENTIFICATION, AND STUDIES ON THE METABOLISM OF RUMEN MICRO-ORGANISM GROWTH FACTORS PRESENT IN NATURAL MATERIALS

DISSERTATION

Presented In Partial Fuirillraent of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University

By

BURK ALLYN DEHOR ITT, A.B., M.S.

The Ohio State University

1957

Approved by:

Adviser Department of Agricultural Biochemistry ACKNOWLEDGMENTS

I would like to express ray deepest appreciation to Dr. Alvin L. Moxon and Dr. Orville G. Bentley for their guidance and many helpful suggestions during the per­ formance of ray research work. Their interest and leader­ ship is gratefully acknowledged.

The timely suggestions and interest of Dr. Ronald

R. Johnson are greatly appreciated, in addition to his aid in the preparation of Figures 10, 11, and 12.

Appreciation is also extended to the Ohio Agricultural

Experiment Station for providing facilities and financial assistance during the course of this work.

I am indebted to ray wife for her unceasing encour­ agement and cheerfulness throughout the performance of this work, and her aid in the preparation of this manu­ script.

ii TABLE OP CONTENTS

General Introduction

Part I: Isolation and .identification of Compounds Prom Autolyzed Yeast, Alfalfa Ileal, and Casoin iiydrolysa.te wit h. Cellulolytic Factor Activity for Rumen Micro-organisms In Vitro

Literature Review

Experimental Procedures

In Vitro Rumen ^Fermentation Technique

Fractionation Procedures

Dowex - 50 I o n Exchange Resin

Charcoal Treatment

Preparative Scale Paper Chromatography

Elution of the Chromatograms

Chromatographic Identification of Amino Acids

Results Autolyzed Yeast

Casein Rydrolysate Alfalfa Extract

Known Amino Acids Substrate Level Studies

Additive Effects Discussion

Summary Part II: Studies on the Metabolism of the isolated Growth Factors (Valine, Proline, Leucine, and Isoleucine) Literature Review Experimental Procedures Amino Acid Analysis In Vitro Rumen Fermentation Technique Using Valine-l-Clh Determination of «<-KetoisovalerIc Acid Results Investigation of Added Amino Acids Rumen Fermentation (In Vitro) with Valine-l-dU

Isolation of Radioactive -Ketoisovaleric Acid

Cellulolytic Factor Activity of Possible Intermediates in Valine, Proline, and Leucine Metabolism Discussion Summary Literature Cited LIST OP TABLES Table No. Page No 1 * Composition of the Basal Medium Used for the ill Vitro Rumen Fermentation 13 2. Cellulolytic Factor Activity of the C.F. of D - 50 Eluate of Autolyzed Yeast After Separation by Paper Chromato­ graphy with Q0% Phenol 21

3. Cellulolytic/ Factor Activity of the Separated Components From T.P.P.C. of Autolyzed Yeast 2l± If.. Cellulolytic Factor Activity of Casein Hydrolysate and the T.P.P.C. Prepared From Casein Hydrolysate 27

5. Cellulolytic Factor Activity of the Fractions Obtained from Alfalfa Extract #1 by Treatment with Dowex - 50 30 6. Cellulolytic Factor Activity of the Amino Acids Valine, Proline, the Leucines and y-aminobutyric Acid at Varying Levels; Studies on the Additive Effects of These Amino Acids 35 7. The Additive Effect of the Cellulolytic Factor Activity of Valeric Acid, the Amino Acids and Yeast Extract I4.0 8. Study on the Additive Effect of Valeric Acid with Valine and Proline if.2

9. Amino Acid Composition of Fermentation Mixtures 62 10. Cellulolytic Factor Activity of D-Valine, DL-Valine and L-Valine Alone and In Combination with L-Proline 65 11. Amino Acid Composition of Fermentation Mixtures 66 12. Decarboxylation of DL-Valine-l-ClU by Rumen Micro-organisms In Vitro 69

v vi Table TTo. Page No.

13. Relationship of L-Valine andcw-betoiso­ valeric Acid in the in Vitro Rumen Fomentation 80 lij. Cellulolytic Factor Activity of Proposed Intermediates in the Metabolism of Valine and Proline 82 15. Cellulolytic Factor Activity of Proposed Intermediates in the Metabolism of Leucine and Proline 81j. 16. Time Study of Valine and Leucine Plus Proline Versus Isobutyric Acid and Isovaleric Acid Plus 5 -Aminovaleric Acid 86

17. The Cellulolytic Factor Activity of Ornithine 88 LIST OF FIGURES

Figure No. Page No.

1. Diagrammatic representation of the chromato­ grams obtained by preparative scale paper chromatography 17

2. The cellulolytic factor activity in vitro of yeast extract and the fractions pre­ pared from this extract 20

3. Diagrammatic sketch of the chromatographic separation of the amino acids from yeast extract In the top portion of the phenol chromatogram (T.P.P.C.) 22

ll. The cellulolytic factor activity in vitro of hydrolyzed casein and the fractions of hydrolyzed casein prepared by large scale paper chromatography 28

p. The cellulolytic factor activity in vitro of alfalfa extract # 2 , and the prepared fractions from this extract 3 1

6 . The cellulolytic factor activity in vitro of the charcoal filtrate of Dowex - £6 eluate of alfalfa extract # 2 and the fractions of this filtrate prepared by large scale paper chromatography 32

7. The cellulolytic factor activity in vitro of the amino acids valine and proline 3k

8 . Additive effects of the amino acids valine, proline, the leucines and Y-aminobutyric acid as cellulolytic factors in vitro 37 9. The effects of varying cellulose substrate levels upon cellulose digestion in vitro 3 9 10. Diagrammatic separation of the amino acids from flasks with added valine and proline determined at 1 3 , 2l± and 30 hours, and of proline, determined at 13 hours 67 vii v i . i l

Figure Wo. Page No.

1 1 . Tracings of the chromatographic separation of the 2 , l^-dini trophenylhydrazine deriva­ tives obtained from a fermentation with DL-valine-l-ClU, and the corresponding graph of its radioactivity as measured with a gas-flow chromatographic scanner 77 12. Proposed pathways in the metabolic breakdown of valine and proline 90

H’i General Introduction

The significance of rumen micro-organisms, in the utilization of cellulose and other carbohydrates as energy sources by cattle and sheep, was first recognized at about the turn of the twentieth century. As the role of these micro-organisms became better understood, the relationship between their nutritional status and their ability to digest cellulose, synthesize vitamins, and convert non­ protein nitrogen to protein nitrogen became of both prac­ tical and academic interest. Of primary interest, was the determination of the nutritional requirements of this mix­ ed microbial population. Studies at the Ohio agricultural Experiment Station on crude fiber and cellulose digestion in vivo, suggested that certain natural feedstuffs supplied unknown nutri­ ents or growth factors which are essential for cellulose digestion by rumen micro-organisms. This led to the development of an artificial rumen technique, whereby the rumen micro-organisms were cultured in vitro under simulat­ ed rumen conditions. The experimental results obtained by this technique correlated well with previous in vivo results, hence a major contribution had been made toward the determination of the nutritional requirements of these microorganisms. A number of excellent preliminary studies were performed on the quality of roughages, urea utiliza- 1 2 tion, mineral requirements, and the ability of certain natural feedstuffs to enhance the digestion of purified cellulose.

As the major nutritional requirements of the rumen bacteria became known, these nutrients were added to the basal medium used for culturing the micro-organisms. The problem soon narrowed itself to the point where the major­ ity of the nutritional requirements of these micro-organisms had been established, except that the rumen micro-organisms required an unknown nutritional factor or factors before any marked degree of cellulose digestion was obtained. These unknown cellulolytic factors , present in the supernatant of rumen juice, were investigated over a period of several years in this laboratory before their identity was estab­ lished. However, subsequent experiments revealed that these cellulolytic factors were not identical to those responsible for the activity of yeast extract and other natural materials. Hence, the occurrence of other unknown compounds capable of stimulating cellulose digestion by rumen micro-organisms was established, and the experimental studies reported herein are concerned with the identifica­ tion of these cellulolytic factors.

1. Throughout this dissertation, the term cellulolytic factor will be used to designate any material or compound which possesses the ability to enhance the digestion of cellulose by rumen micro-organisms in vitro. Part I Isolation and Identification of Compounds From Autolyzed Yeast. AlfalfaT'Meal, and Casein' Hydrolysate with Cellulolytic factor Activity For Rumen Micro-"" orff&ni'ams In Vitro

Literature Review

The existence of specific nutritional factors supplied by natural feedstuffs, which are required by rumen bacteria for the efficient digestion of cellulose, I'ras first postulat­ ed by Burroughs £t al^ (li|) in 191+9. The results of his in vivo roughage digestion trials (1 1 ; 1 2 ,* 1 3 ;111) indicated that these factors are associated in nature with roughages and protein-rich feeds. In one study (11), rumen bacterial counts were made, and bacterial numbers were observed to increase in direct proportion to roughage digestion, in an effort to gain more detailed information on the factors in­ volved in roughage digestion by rumen micro-organisms,, an in vitro artificial rumen technique was developed by Burroughs

(1 0 ), somewhat similar to that described by Marston (ipO). The objective of this study was to culture the rumen micro­ organisms under controlled conditions in the laboratory, where the nutrition and action of these micro-organisms could be closely observed. Basically, this method involved growing the rumen micro-organisms on a purified medium for

36 hours, and then transferring half of this material to a second flask containing another aliquot of nutrients, there- k by diluting out the required nutritional factors from the inoculum. Various materials were added to the medium to determine their effect on the rate of cellulose digestion. Autoclaved rumen fluid and an autoclaved water extract of manure proved beneficial. In further in vitro studies * * (1 5 ), good and poor quality roughages were tested as sources of cellulose. Available nitrogen, a complex mineral mixture, and an autoclaved water extract of cow manure were found to enhance digestion of poor quality roughages (corn-stover, wheat straw, corncobs, and mature timothy-blue grass hay), and filter paper cellulose. Addition of these supplements failed to increase cellulose digestion with the good quality roughages (clover hay, rye hay, and alfalfa hay). In vitro

studies on some common cattle feeds (1 6 ) indicated that dried distillers solubles, soybean oil meal, linseed oil meal, cane molasses, corn wheat bran, and cottonseed meal enhanced cellulose digestion, while meat scraps, fish meal, liver meal, and oats had no influence. After the initial reports by Burroughs and Marston, a number of investigators became interested in this problem, and research was initiated at several institutions on the Isolation and identification of these growth promoting sub­

stances for rumen bacteria. Ruf et al. (ii7 ), using the in vitro artificial rumen technique as described by Arias

et al. (2 ), found that a number of natural materials stimu- 5 lated cellulose digestion (alfalfa meal, yeast, cow manure extract, etc.). They attempted to isolate the growth factor activity from yeast extract and cow manure extract, and their results indicated that the activity was not mineral or protein in nature, could be adsorbed on Norite and eluted with acetone or ethanol, and was heat stable. Further experiments also indicated that the factor was apparently not an amino acid or a B-complex vitamin, how­

ever, hall at al. (2 8 ) found that vitamin B.^ and biotin enhanced cellulose digestion with washed suspensions of rumen micro-organisms, but not as much as the degree of stimulation obtained from yeast extract. Meanwhile, Hungate (35)* working with a number of cellulose digesting micro-organisms isolated from the rumen, found that most of them required rumen fluid in the medium, and that yeast extract could be substituted for rumen fluid

with certain types of rumen bacteria. Bryant (8 ) also utilized rumen fluid in his basal medium for the isolation

of micro-organisms from the rumen. Doetsch at al. (2 1 ) have observed that rumen fluid contains unknown substances essential for optimal growth of some rumen bacteria. These substances were not provided by a commercial medium devised for nutritionally fastidious bacteria. Quinn (1|1{.) (as cited by hershberger) (32), working with.a thermophilic cellulose - digesting micro-organism, fractionated yeast extract for cellulolytic factor activity and observed that the active fraction was water soluble, adsorbed on Norite at pH 3>

but not at pH 6 , 8 , or 10, and was destroyed by ashing. Wasserman et al. (56) reported that isolated cultures of rumen bacteria required or were stimulated by certain vitamins, purines, and pyrimidines. The basal medium employed in his studies contained yeast extract. Using the sediment obtained from the high speed cen­ trifugation of whole rumen juice as inoculum in an in vitro fermentation, Bentley et al. (i|;6 ) observed that autoclaved rumen juice supernatant, hot water extracts of various plant materials (alfalfa, ladino clover, etc.), molasses, dried distillers solubles, and yeast extract markedly in­ creased the rate of cellulose digestion. Biotin, vitamin

1*1 2 * PABA, xanthine, uracil, guanine, and adenine improved cellulose digestion when added to the basal medium but not to the degree observed with rumen juice supernatant, water extract of alfalfa, yeast or molasses. Rumen juice was fractionated by treatment with lead acetate, and the activity remained in the supernatant. When the dried supernatant was extracted with absolute alcohol, the activity was in the absolute alcohol fraction. The activ­ ity could be adsorbed on Norite at pH 3 and but not at pH 5, 6 , or 9-11* and could be eluted with dilute KOH or E*fcOH:H 0: Nh Oh 1) * The cellulolytic factors, as 2 Zx obtained by them from rumen juice, were soluble in butanol, propanol, methylcellusolve, water and ethanol, and relative­ ly insoluble in acetone and ether. The active factors in rumen juice and alfalfa extract were also found to be stable to heat. Autoclaving at 15 lb. pressure for as long as 21+ hours in neutral or alkaline solution (IN NaOh) did not destroy the activity, however, some activity was destroyed after autoclaving for 1+ hours in IN HC1, and a

complete loss of activity was observed after 21+ hours. At this same time, McNeill £t al. (l+l;l+2) were attempting to fractionate rumen fluid for factor activ­ ity as measured by bacterial counts of rumen micro-organisms

incubated at 39°C for 72 hours. They found that the activ­ ity could be extracted by adsorption and elution methods on Norite, but that these factors were selective on the organisms. A little different type of observation was reported by Garner at al. (27) in 1951+* They found that in extracts of bovine and ovine rumen fluids, feeds fermented by rumen organisms, and in some types of silage there were factors which inhibited the growth of Pseudomonas aeruginosa and Aerobacter aerogenes, two organisms isolated from animals with rumen dysfunction. These extracts also enhanced cellulose digestion in vitro, using washed rumen cells from healthy animals. Thus, Garner et al. suggest that this factor not only enhances the growth and function of desirable rumen organisms, but inhibits the growth of organisms which sometimes contaminate the rumen. Since these unidentified factors were water soluble and often occurred in largest quantities in feeds either rich in protein or in natural feeds rich in non-protein nitrogen, hall £t al. (29) attempted to alter water in­ soluble proteins in such a way as to produce these stimu­ latory factors. Purified casein was subjected to a mild hydrolysis either by acid, alkali, or enzymatic methods, and in each instance the mildly hydrolyzed casein exhibited a highly favorable influence upon a washed suspension of rumen micro-organisms in the digestion of cellulose. Complete hydrolysis resulted in the complete loss of such activity, suggesting that the unidentified active factors were water soluble peptides. Partial hydrolysates of soy­ bean protein, chicken feathers, hair, and blood meal also showed factor activity. In 195k-, Bentley et al. (5) reported that a volatile fraction from rumen juice had cellulolytic factor activity. Distillates obtained from acidified rumen juice (pH 2.0 to

2 .5 ) were active, whereas distillates from basic rumen juice (pH 10 to 11) were inactive. When the residue in the distil­ lation flasks was neutralized and added to the fermentation flasks, the alkaline residue was active while the acid residue showed no activity. These results suggested to

tne authors that the cellulolytic factor or factors could

be a steam volatile fatty acid. Upon testing the pure

volatile fatty acid3 a3 cellulolytic factors, they found

valeric and caproic acids to be quite active. iso-valeric

and iso-butyric acids also increased cellulose digestion,

but not quite as markedly as the normal and acids. They found no activity in formic, acetic, propionic, butyric, and the Gy to straight chain fatty acids. It had been suggested previously by El-Shazly (2l\.) that rumen micro-organisms formed branched chain five carbon acids from amino acids and proteins. Thus, a study of the -amino homologues of the active volatile fatty acids was made, and it was found that the amino acids valine and proline increas ed the digestion of cellulose. Later in a complete report, Bentley e_t al_. (3) analyzed the volatile fatty acid fraction from rumen juice and found that of the cellulolytically active acids present, valeric acid was the major constituent Valeric acid was also observed to increase the quantity of trichloroacetic acid insoluble protein in the fermentation flask {an estimate of bacterial growth). The importance of these acids in rumen bacterial nutrition was further siibstantiated by the work of Bryant and Doetsch (9). Work­ ing with Bacteroides succinogenes, a cellulose digesting bacterium isolated from the bovine rumen, they found that 10 this organism required a combination of a branched chain fatty acid (i30-butyric, iso-valeric, or DL-*-methyl-n- butyric acid) and a straight chain acid (valeric or caproic acid) for growth on a purified medium. Upon the basis of the observation that the cellulolytic factor activity of alfalfa meal, yeast extract and dried distillers solubles could not be steam distilled from acid solution, Bentley erfc sdL. (3) postulated the existence of additional growth factors. Because certain free amino

acids were active a 3 cellulolytic factors, they suggested that the activity of these materials might reside in their free amino acid content. Working with partial acid hydrolysates of chicken feather meal, casein, and gelatin, hall et al. (3 0 ), had found that the factor activity of these materials was non­ volatile from acid or alkaline solution by steam distill­ ation.

The object of the present 3 tudy was to isolate and identify the cellulolytic factors of autolyzed yeast, hot water extracts of alfalfa meal, and casein hydrolysate. Prom the previous work it appeared that the factor activity contained in these materials was not one of the volatile short chained fatty acids, and thus experimental work was begun by investigating that fraction containing the amino acids and water soluble peptides. Experimental Procedure

In Vitro Rumen Fermentation Technique The in vitro rumen fermentation technique used for these studies was essentially the same as previously re­ ported from this laboratory by Bentley et al. (3). Inoc­ ulum was obtained from a steer fitted with a permanent ruminal fistula and maintained on a ration of good - quality alfalfa hay, and the rumen juice was expressed from the ingesta by filtering through cheesecloth with the aid of a large fruit press. One liter of this rumen juice was centrifuged in a Sharpies supercentrifuge, and the sediment collected on a celluloid liner inside the centri­ n g © cylinder. Since rumen juice contains plant debris and protozoa in addition to bacteria, an attempt to minimize these sources of possible contamination was made by discarding all of the sediment within one inch of the bottom of the celluloid liner. All of the sediment above this portion was suspended in 250 ml. of phosphate buffer (pH 7*0), with the aid of a loose-fitting Potter - Elvehjem homogenizer. The buffer consisted of 1.059 g. of Na£HP0^, 0.1^36 g- of K ^ P O ^ and 100 mg. of cystelne-HCl per liter.

Ten ml. of this suspension, which was equivalent to JLj.0 ml. of whole rumen juice, was used to inoculate each in vitro fermentation flask.

11 The composition of the basal medium U 3ed is given in Table 1. The fermentation w&s carried out in 175 ml. wide mouth bottles, the total volume of the mixture being ad­

justed to 1 0 0 ml. at the end of the fermentation period. Temperature of the flasks was maintained at 39 + 0.5°G in a thermostatically controlled water bath. Each flask was gassed individually with GO£, by separate leads from a

common manifold connected to a large CO2 cylinder. The

CO2 served to maintain anaerobic conditions and provided a degree of agitation. The physical apparatus was so designed that twenty-four fermentation flasks could be

accommodated at one time. At approximately 2, 8 and 2ij. hour after the start of the fermentation, pH determinations were made, and the pH adjusted to 6.9 with a saturated solution of NagCO^, when required.

Unless otherwise designated, the fermentation was

carried out for a 30 hour period. At the end of this time,

the volume of the flask was brought to 100 ml. with dis­

tilled water, the contents thoroughly agitated to suspend

the insoluble material (cellulose and bacterial cells),

and duplicate 10 ml. aliquots taken to determine the

amount of cellulose digested. Cellulose determinations were made by the method of Hershberger et al. (3k), which

is a volumetric method developed in this laboratory, and based on the Crampton and Maynard procedure (20). 13

Table 1 Composition of the Basal Medium Used For the In Vitro Rumen Fermentation

Constituent Grams/100 ml. Cellulose0-...... 1.00 Glucose...... 0.10 Urea...... 0.126 Na CO ...... 0.20

Na2JtiP0^D...... 0 .1 1 3 NaH PO,...... 0.109 2 1+ KOI...... 0.01+3 NaCI...... O.Oi+3

MgCO^...... 0 .001+

CaCl2 ...... 0 .001+ Na SO ...... 0.015 i+ FeC1 ^.6 H^ 0 ...... O.OOkk Bio tin...... 20 gamma FABA...... 50 gamma

Sediment0 ...... 10 ml. a. Solka - Floe lj.0A - Brown Co., Berlin, New Hampshire b. All of the minerals except Na CO , FeCl and CaCl were added Prom a single stock solution. 3 3 2 c. The sediment was obtained as previously described in the text. 14 Fractionatlon Procedures

Dowex - 50 Ion exchange re3 In — Hot water extracts of alfalfa meal and aqueous solutions of autolyzed yeast were initially fractionated by passage through a bed of Dowex - 50 ion exchange resin. The Dowex - 50 was placed in a Buchner funnel and washed with a 25^ NaOli solution, using suction, to remove any alkali displacable impurities. The resin was then converted to the H+ form by washing with ijN HC1 until the filtrate was distinctly acid. Next, the resin was washed repeatedly with distilled water until the filtrate became neutral, indicating that all of the excess acid had been removed. The resin was then ready for use. The extract was poured onto the resin bed, and the bed washed repeatedly with distilled water to flush any material not adsorbed by the resin into the filtrate. This solution shall be referred to as the Dowex - 50 filtrate (D - 50 filtrate) in the following sections. The D - 50 filtrate was found to contain only a minute trace of ninhydrin pos­ itive material. The resin bed was then washed with a $0% NH^Oh solution until all of the ninhydrin positive material had been eluted, as determined by spotting successive por­ tions of the eluate on filter paper, drying and spraying with ninhydrin. Removal of ammonia from this eluate was accomplished by boiling. This eluate will be called the Dowex - 50, 50% NH^OH eluate (D - 50 eluate). 15 Charcoal (Darco G-60) treatment — The Dowex - 50, 50%

Nhj^OH eluate was purified further by passage through a charcoal (Darco G-60) bed in a Buchner funnel. The char­ coal bed was then washed with distilled water until the filtrate was free of ninhydrin positive material. A rather large volume of distilled water in relation to the sample volume was required for this procedure. This fil­ trate will be referred to as the charcoal filtrate of

Dowex - 50, 50% Nh^Oh eluate (C.F. of D - 50 eluate). Preparative scale paper chromatography — The C.P. of D - 50 eluate was further fractionated by large scale unidimensional ascending paper chromatography. A large wooden balance cabinet (36" high x 3 6 " wide x 1 8 " deep) with glass panels on the front, top and both sides, was used for a chromatographic chamber. The entire front panel of the cabinet slides upward allowing easy access to the chamber and the grooves are backed by felt to de­ crease evaporation. The wooden surfaces exposed on the interior of the cabinet were covered with paraffin, to decrease absorbtion of the solvents. Glass rods served as supports for the paper chromatograms. Whatman #1 filter paper sheets (1 3 " x 22|r") were used for chromato­ graphic separation of the C.P. of D - £0 eluate which was applied as a streak along the base line. The solvent system utilized for this separation was 8 0 % phenol - 2 0 % 16 water, and was placed In a large porcelain pan at the bottom of the chamber. The chromatograms were developed for 36 to i{.8 hours at room temperature. Slution of the chroma to grams — The developed chromato­ grams were removed from the chamber, air dried overnight in a hood, and then sprayed with a large quantity of ether to remove any residual phenol or phenol decomposition products. After allowing the ether to evaporate, the chromatograms were dried in an oven at 1 0 5 °C for 15 minutes. Upon removal of the paper sheets from the oven, the separated amino acids were visible as fluorescent zones under ultraviolet light. It was found that a non-fluores­ cent zone consistently appeared approximately between the R^, values 0.60 and 0.65. Figure 1 gives a diagrammatic picture of the chromatograms obtained. No differentiation between the components, except for the non-fluorescent zone, Is probably a result of overloading the chromatograms. The chromatograms were divided at this non-fluorescent zone into two portions, and the material from each portion was eluted with hot water. The solution obtained from that portion of the chromatogram between the non-fluorescing zone (Rf 0 .6 0 - 0 .6 5 ) and the solvent front was termed the top portion of the phenol chromatogram (T.P.P.C.), while the eluate from the zone to the base line wa3 called the bottom portion of the phenol chromatogram (B.P.P.C.). 17

13*

SOLVENT FRONT

NON-FLUORESCING ZONE Rf- 0.60 •i M OJ

ap.p.c.

i r ORIGIN

I

Figure 1. Diagrammatic representation of the chromatograms obtained by preparative scale paper chromatography T.P.P.C. (top portion of the phenol chromato­ gram) ; B.P.P.C. (bottom portion of the phenol chromatogram) 18 These solutions were concentrated by boiling before being tested in the artificial rumen. Chromatographic identification of amino acids — The amino acids from those fractions showing cellulolytic factor activity were identified by unidimensional ascending paper chromatography. A number of the well known amino acid

developing solvents were used for this purpose (7 )-(1 ) Butanol:Acetic Acid:Water (lf:l:l); (2) Pyridine;tertiary Amyl AlcoholiWater (35:35:30); (3) secondary Butanol: 3% NH^O-ti (3:1); (4) Phenol:Water (80:20). Leucine and Isoleucine were separated with tertiary Amyl Alcohol: Propanol:Water (lj.:l:l) (18). One further solvent sys­ tem, developed by the author, was found very useful for the separation of amino acids. This solvent system was composed of Ethanol:Butanol;Pyridine:Water (60:10:5:25). The chromatograms were dried, sprayed with ninhydrin reagent, and color of the- spots was developed by heating in an oven. Since values show a wide fluctuation, the unknown amino acids were identified by comparing the suspected known, the unknown, and a mixture of the suspected known and unknown amino acids on the same chromatogram. This technique compensates for any deviation of the value in the unknown caused by extraneous material. Repeatable results, by this technique, in three to four different solvents constituted identification. Results

Autolyzed Yeast (Dlfco yeast extract) The results of the preliminary fractionation of yeast extract (Dowex - 50 and charcoal treatment) are summarized in Figure 2. These curves constitute the average results obtained from two in vitro rumen fermentations. It Is clear that the cellulolytic factor activity of yeast extract is present in the Dowex - 50, 50% NH^OH eluate, and the charcoal filtrate of this eluate. The cellulolytic factor activity of the fractions of C.F. of D - 50 eluate obtained by paper chromatography, using 80% phenol as a solvent, is shown in Table 2. These results indicate that the major portion of the cellulolytic factor activity was found in

the top portion of the phenol chromatogram (above 0 .6 5 ). When the extract of the top portion of the phenol chromato­ grams was analyzed chromatographically with 80% phenol as the solvent system, three amino acid spots were found. Chromatography in other solvents, as can be seen In Figure 3, consistently showed four amino acids to be present. These amino acids were identified as valine, proline, the leucines (both leucine and Isoleucine were found to be present, using the tertiary-amyl alcohol:propanol:water solvent), and t- aminobutyric acid. Subsequent work with two-dimensional chromatography showed that valine and lf-aminobutyric acid possessed the same R-^ value in 80% phenol.

19 20

8 0

v> 6 0 uj

3 -J 2 0

1.0 2.0 3.0 4.0 ML. YEAST EXT. (Iml.*50mg.)

!

Figure 2. The cellulolytic factor activity in vitro of yeast extract, and the fractions prepared from this extract (A) Yeast extract; (B) Dowex - 50, 50% IJh^OJi eluate of yeast extract; {C ) Dowex - 50 filtrate of yeast extract; (D) Charcoal filtrate of the Dowex - 50, 50^ Wh^Oh eluate of yeast extract 21

Table 2 Cellulolytic Factor Activity of the C.F. of D - 50 eluatea of Autolyzed Yeast After Separation By Paper Chromatography With 80% Phenol

Additions to Cellulose Digestion, % Basal Medium 3xp. 1 Exp. 2 Exp. 3 3/12/56 l|/3/56 5/1/56 5/8/56

None 7.7 21]..7 8.8 10.0 13.1+ Valeric Acid, 30 mg. 61.0 59.8 71+.6 62.1 5k-2

C.F. of D - 50 eluate® • • • • 61.0 • • • • 58.7 1+2.9 T.P.P.C.b 65.5 63.2 63.2 lj.6.2 58.7

B.P.P.C.c 12.8 31.5 8.8 Ik-5 • • • •

a. Charcoal filtrate of Dowex - 50, 50% Nil],Oh eluate. The quantity utilized was equivalent to the amount of chromatographic eluate used. b. Top portion of phenol chromatogram (above R^. 0.65)• c. Bottom portion of phenol chromatogram (below 0.60)• 22

§ 3 O' 0* 0 ' 0 * O 3 02 0 4 0* 04 0 3 8;

TRRC. T.P.P.C. T.P.P.C. T.P.P.C. 8 0 % PHENOL PYR.U-AmOH: BuOH:HAc: EtOH:BuOH: h2o h2o PYR.: h£o

M.EUCINES; 2 = VALINE; 3=PROLINE; 4=»-NH2-B.A.

Figure 3. Diagrammatic sketch of the chromato­ graphic separation of the amino acids from yeast extract in the top portion of the phenol chromato­ grams (T.P.P.C.) Solvents: (1) 80% Phenol = Phenol-.Water (80:20); (2) Pyr: t-Am0ii:±i20 = Pyridine: tertiary Amyl Alcohol Water (35:35:30); (3) BuOh:hAc:hgO = Butanol:Acetic Acid:Water (1{.:1:1); (J|) 2t0h:BuOn: Pyr:hgO = Ethanol Butanol:Pyridine:Water (60:10:5:25) 23 Enough of this top portion of the phenol chromato­ grams (T.P.P.C.) was prepared to carry out a second pre­ parative scale separation, utilizing differont solvents. Two separations were made, the first with pyridine: tertiary amyl alcohol: ti^O (35:35>:30) as the developing solvent, and the second using secondary butyl alcohol: 3% NHj^Oh (3:1). After development these chromatograms were divided into three sections and each section eluted with hot water. The amino acid composition of the three eluates was determined by paper chromatography, and similar identifications were obtained from both separations. Frac­ tion I contained the fastest moving component, which was found to be the leucines (leucine and isoleucine). Fraction II contained the middle two amino acids which were valine and proline, while fraction III wasY-aminobutyric acid. The fractions thus obtained were then tested for factor activity in the in vitro rumen fermentation, and the results are shown in Table 3* The T.P.P.C. used for the separation by pyridine: tert.-amyl alcohol: I^O was the same one used in experiment 2, Table 2. Separation by sec.-butyl alcohol: 3% NH^OH was made using the same T.P.P.C. as reported in experiment 3 of Table 2. No explanation for the low cellulose digestion of the T.P.P.C. in this second experiment (5/3/56) of Table 3 can be made, but the data presented in Table 2 and Table 3

Cellulolytic Factor Activity of the Separated Components From T.P.P.C.a of Autolyzed Yeast

Additions to Cellulose Digestion, % Basal Medium 1+/16/56 5/3/5b

None 12.2 I4-0 Valeric Acid, 30 mg. Controls 61.0 Controls liO.6 C.F. of D-50 Eluate*3 37.2 62.0 T.P.P.C. 3 9 4 19.0

Fraction lc , 29.2 3^.9 Fraction 11°- R e chroma t o graphing 33.8 Rechromatographing ij.9.6 Fraction llle Solvent-pyridine: 8.8 Solvent-sec-butyl 4 - 5 Fraction 1 plus 11 tert-amyl alcohol: alcohol: 3% 14-5.1 Fraction 1 plus 111 e 2o 3 9 4 Fraction 11 plus 111 28.1 31.5 a. Top portion of phenol chromatogram (above Rf 0.65). b. Charcoal filtrate of Dowex - 50, 50% Nh^On eluate. The quantity utilized wa3 equivalent to the amount of chromatographic eluate used. c. Leucines. ) d. Valine and proline. ) Identified by paper chromatography. e. Y-aminobutyric acid.) 25 the activity of the separated components of this solution, clearly indicate its activity. • Occasionally this lack of cellulose digestion for no apparent reason is observed. It can be concluded from the results presented In Table 3 that the cellulolytic factor activity of T.P.P.C., and thus of yeast extract, lies in the amino acids valine, proline and the leucines. Experiments utilizing these known amino acids as cellulolytic factors in the in vitro rumen fermentation are presented at the end of this section.

Casein iLydrolysate Commercial casein hydrolysate (acid) was used as a cellulolytic factor source for these experiments. Because of the relative purity of this material, It was believed that preliminary purification with Dowex - 50 Ion exchange resin and charcoal, as used with yeast extract, was not necessary. The casein hydrolysate was applied directly to the filter paper sheets and chromatographed in 80% phenol. The chromatograms were divided into two sections, at approximately Rf 0.65> and the two sections were eluted with hot water. These eluates (T.P.P.C. and B.P.P.C.) were then passed through charcoal to remove any phenolic materials which may be toxic to rumen micro-organisms. As before, the T.P.P.C. and B.P.P.C. were concentrated and tested for factor activity in the in vitro rumen fermenta­ tion. In a preliminary experiment, In which only enough 26 material for one trial was prepared, the B.P.P.C. was lost in the concentration procedure and thus only data for the T.P.P.C. was obtained. These results are given in Table [}..

A second complete experiment was then carried out, and the results are shown in Figure ij.. The curves shown are a summary of two separate experiments, and the results are in agreement with the data presented in Table ij.. The cellulolytic factor activity of casein hydrolysate was found to be entirely in the T.P.P.C., while the B.P.P.C. showed a slight degree of inhibition. Paper chromatographic analysis of the T.P.P.C. of casein hydrolysate revealed the amino acids valine, proline, the leucines and trace amounts of an unknown amino acid. From available R^. values in a number of solvents, it is believed that this unknown material may be methionine sulfoxide. An inhibition of cellulose digestion was observed when levels of casein hydrolysate exceeding 100-150 mg. per flask were used. Whether this inhibition is caused by an excess of certain amino acids, or is simply a result of an excess of all the amino acids, is not known.

Alfalfa Extract Hot water extracts of alfalfa meal were prepared by mixing the ground alfalfa with ten parts of water, bringing 27

Table k

Cellulolytic Factor Activity of Casein Hydrolysate and the T.P.P.C? Prepared From Casein Hydrolysate

Additions to Gellulose Basal Medium Casein (mg. Digestion, %

None --- 13.il- Valeric Acid (30 mg.) --- 6it.ii.

Casein Hydrolysate 10 3 2 .6

Casein Hydrolysate 25 5*1.2 Casein Hydrolysate 5o 63.2

T.P.P.C.a of Casein Hydrolysate 10 214-.7

T.P.P.C. of Casein Hydrolysate 25 38.3 T.P.P.C. of Casein Hydrolysate 5o 56.lt

a. Top portion of the phenol chromatogram. b. The weight of casein, in mg., equivalent to the amounts of material tested. 28

80

z o • A £ 6 0 Ul 2 o Ul 4 0 (O o _i

20 LU O , -o C

10 20 30 40 50 MG. OF HYDROLYZED CASEIN

Figure The cellulolytic factor activity in vitro of hydrolyzed casein and the fractions of hydrolyzed casein prepared by large scale paper chromatography (A) Casein hydrolysate; (B) T.P.P.C. (top portion of phenol chromatogram) of casein hydrolysate; (C) B.P.P.C. (bottom portion of phenol, chromatogram) of casein hydro­ lysate 29 the mixture to a boil, and filtering through a Buchner funnel. The extract was then subjected to the Dowex - $0 rosin and charcoal treatments. Two batches of alfalfa extract were prepared, the first being tested only for the fractionation of its cellulolytic factor activity with Dowex - 50 (see Table 5) • Figure 5 illustrates the cellulolytic factor activity of these various fractions, plus the activity of the charcoal filtrate of D - 50 eluate, for alfalfa extract #2. These response curves are an average of the results obtain­ ed in two separate in vitro fermentations. The T.P.P.C. and B.P.P.C. of the C.F. of D - 50 eluate for alfalfa extract #2 were prepared as previously described, and their cellulolytic factor activity determined. These results are shown in Figure 6, and represent the average of two separate experiments. As was found with yeast extract and casein hydrolysate, the activity of the alfalfa extract occurred in the T.P.P.C. Paper chromatography of this solution revealed the presence of the amino acids valine, proline, the leucines and t-aminobutyric acid. It will be noted in Figure 5 that passage through charcoal of the D - 50 eluate resulted in the loss of a portion of the cellulolytic factor activity. Paper chromato­ grams of these solutions indicated that charcoal treatment of the D - 50 eluate decreased the content of the leucines. 30

Table 5 Cellulolytic Factor Activity of the Fractions Obtained From Alfalfa Extract #1 by Treatment With Dowex - $0

Additions to Equivalent Cellulose Digestion, % Basal Medium to grams 6/21/56 6/28/56 Alfalfa

None -- 13. Ij- 11+.5 Valeric Acid (30 mg.) --- 61+.1+ 68.9 Alfalfa Extract #1 0.5 51.9 -- Alfalfa Extract #1 1.0 55-3 65.5 Alfalfa Extract #1 1.5 1+9.6 ---

Alfalfa Extract #1 2.0 kb-o 61 .0

Dowex - $0 Filtrate 0.5 16.8 -- Dowex - 50 Filtrate 1.0 15.6 27 .0

Dowex - 50 Filtrate 1.5 16.8 ---

H f—1 Dowex - 50 Filtrate 2.0 H • 29.2 Dowex - 50, $0% Nh^Oh Eluate 0.5 38.3 -- Dowex - 50, 50% NhijOh Eluate 1.0 61.0 62.1 Dowex - 50, %0% N±s^0h Eluate 1.5 6I+.1+ --

Dowex - 50, 50% Niij^Qii Eluate 2.0 61+. 14- 7 5 .7 31

80

to 4 0 _i 3 tu o 20

6.0 12.0 18.0 24.0 ML. ALFALFA E X T.(l2ml.S|gm.)

Figure 5» The cellulolytic factor activity in vitro of alfalfa extract #2, and the prepared fractions from this ex­ tract

(A ) Crude alfalfa extract; (B ) Dowex - 50, 50% Nu^Gm eluate of alfalfa extract; (C) Dowex 1 50 filtrate of alfalfa extract; (D) Charcoal filtrate of Dowex - 50> 50^ NHl Oh eluate of alfalfa extract 32

80

o H 6 0 (/> UJ e> o UJ 4 0 (/> o => _J •■J 2 0 UJ o

1.0 2.0 3.0 4.0 ML. ALFALFA EXT. (2ml.*lgm.)

Figure 6. The cellulolytic factor activity in vitro of the charcoal filtrate of Dowex - NhhOii eluate of alfalfa extract #2 and the fractions of this filtrate prepared by large scale paper chromatography (A) Charcoal filtrate of Dowex - 50, 50% Nh^Oh eluate of alfalfa extract; (B) T.P.P.C. (top portion of phenol chromatogram); (C) B.P.P.C. (bottom portion of phenol chromato­ gram) 33 This possibility was investigated by comparing the ratio of the leucines to valine as determined on paper chromato­ grams with a densitometer. The ratio of the leucines to valine in alfalfa extract was approximately 1.0; in D - SO eluate 0.88; and in C.P. of D - £0 eluate 0.21. Apparently the leucines were partially adsorbed by the charcoal, and were not eluted with distilled water.

Known Amino Acids Those amino acids which were found to be consistently present in the T.P.P.C. of the materials studied were tested in the in vitro rumen fermentation for cellulolytic factor activity. Response curves, using various levels of the amino acids, were run to determine the level at which maximum cellulose digestion was obtained. Figure 7 illus­ trates a typical response curve with valine, proline and valine plus proline. From this and similar experiments it was concluded that 6 mg. would be the optimum level for testing the amino acids. Table 6 shows the cellulolytic factor activity of the various amino acids singly and in combination with each other. The data presented in this Table is typical of the results obtained with these amino acids in other experi­ ments. As can be seen in Table 6, % -aminobutyric acid possesses no cellulolytic factor activity, while proline exhibits some activity. Also, V-aminobutyric acid did not 3k

80

• o 60 UJ

ui 4 0

-i 2 0

2 4 6 8 10 t-! MG. OF EACH AMINO ACID

Figure 7• The cellulolytic factor activity in vitro of the ajnino acids valine and proline (A) Valine plus proline; (B) Valine; (C) Proline 3 5

Table 6 Cellulolytic Factor Activity of the Amino Acids Valine, Proline, the Leucines and Y-Aminobutyric Acid at Varying Levels; Studies on the Additive Effects of These Amino Acids

Additions to Cellulose Digestion, 0/ Basal Medium /» Mg. of Added Amino Acid 0 mg. 2 mg. 1* mg. 6 mg.

None 21.3 Valeric Acid, 30 mg. 70.0 Y'-Aminobutyric Acid 28.1 29.2 17.9 Proline (P) 36.0 1*0.6 1*1.7 Valine (V) 38.3 1*2.8 55.3 Leucinesa (L) 1*0.6 1*7.1* 51.9

V plus L 1*5.1 51*.2 56.1* V plus P . 57.6 71.2 71*. 6

L plus P 51*. 2 67.8 76.8

V plus P plus L 58.7 67.8 75.7

a. Leucine and Isoleucine 36 show an additive effect when tested in combination with the other amino acids. Valine and the leucines (half leucine and half isoleucine) show a somewhat greater stimulation for cellulose digestion than proline. When leucine and isoleucine were tested singly for cellulolytic factor activ­ ity, leucine was found to show the greatest stimulation. Since both of these amino acids were found in the materials examined, a mixture was employed for these studies. It is thought that valine and the leucines may function in the same manner as cellulolytic factors, since a mixture of the two did not show an Increase in cellulose digestion. When prollne was added with either valine or the leucines an additive effect was observed. This particular effect is more apparent in Figure 7- A mixture of valine, proline and the leucines gave no further stimulation, again Indicat­ ing that the function of valine and the leucines may be similar. The observations mentioned above are quite readily seen in Figure 8, which is a bar graph of the data, at the 6 mg. level, given in Table 6.

Substrate Level Studies At this point the question arose as to whether an additive effect of these various cellulolytic factors, at their optimum concentration, might be masked by a limiting cellulose substrate. Experiments in which 30 mg. of valeric acid were added at substrate levels from 0 to 3 g., 37

BASAL | v Al e r i c AGID.SOMOT

PROLINE. 6MB. VALINE.6M0 LEUO INES.6MO. 1 VALINE PLUS LEUCINES.6MB. EACH I VALINE PLUS PR0LIHE.6M0. EACH LEUCINES PLUS PROLINE.6 MG. EACH D VALINE PLUS PROLINE PLUS LE U 01 N E S.6 MO. EACH I

10 20 30 40 50 60 70 80 CELLULOSE DIGESTION,%

Figure 8. Additive effects of the amino acids valine, proline, the leucines and - aminobutyric acid (f-NHg-B.A.) as cellulolytic factors in vitro

Ite 38 at 0.5 g* intervals, were performed, and the curves shown in Figure 9 were obtained. The increase in the amount of trichloroacetic acid insoluble nitrogen (a measure of

bacterial growth) as well as cellulose digestion, wa3 measured. From these results, it was concluded that 2 g. of cellulose substrate would be better suited for further studies. To facilitate measurements on the amount of cellulose digested using 2 g. of substrate, the volumetric method of cellulose determination, developed by Hersh­ berger ejb alL. (3k.) in this laboratory, was accordingly expanded to cover this level.

Additive Effects The data in Table 7 gives the results obtained on the additive effect of valeric acid, the amino acids and yeast extract. Two grams of cellulose substrate were used, and the digestion is reported in the Table as grams of cellulose digested rather than percent digestion. Grams of cellu­ lose digested seems to 'be preferable, since with 2 g. of cellulose the percentage digestion could be less than with one gram of cellulose, while the actual amount of cellulose digested would be larger. The quantity of each material used was considered to be enough to give maximum cellulose digestion. From the results obtained, it appears that the cellulolytic stimulation from the individual materials is not significantly different from the stimulation obtained 39

20.0

aso

12.0

6.0

I Q20| 4.0 A A MG. OF TCA INSOLUBLE-

0 Q5 1.0 1.5 20 2 5 GRAMS OF CELLULOSE 5UB5TRATE

Figure 9. The effect of varying cellulose substrate levels upon cellulose digestion in vitro Table 7

The Additive Effects of the Cellulolytic Factor Activity of Valeric Acid, the Amino Acids, and Yeast Extract

Grams Cellulose Digested Additions to Basal Medium Exp.#l Exp.#2 Exp.#3 Ave.

None 0.323 0.189 0.336 0.283 + 0.06

Valeric Acid (V.A.), 30 mg. 0.871 0.885 0.801+ 0.853 + 0.03

Amino Acid (A.A,)a 8 mg. each 0.831 0.761+ 0.871 0.822 + 0.01+

Yeast Extract (Y.E.), 250 mg. 0.81+5 0.778 1.005 0.876 + 0.09

V.A. plus A.A, 0.992 0.791 0.952 0,912 + 0.08

V.A. plus Y.E. 0.81+5 0.751 1.112 0.903 + 0 .11+

A,A, plus Y.E. 0.912 1.058 1.112 1.027 + 0.08

V.A. plus A.A. plus Y.E. 0.898 1.005 1.153 1.019 + 0.09

a. The amino acids valine, proline and the leucines. k l with the various combinations. To study this additive effect further a standard valeric acid curve was run, with concentrations of valeric acid from 0 to 30 mg. at £> mg. intervals. A second series of flasks was also run to which 6 mg. each of valine and proline were added at each level of valeric acid. The results of this experiment are given in Table 8. A slight stimula­ tion of cellulose digestion was observed at the lower valeric acid levels (5 and 10 mg.) from the added valine and prollne, but at the higher levels there was no further stimulation from valine and proline. Since there was no additive effect at the level of valeric acid giving maximum digestion by itself, it was concluded that these factors are not additive. The cellulolytic factor activity of valine, proline and the leucines, alone and in combination with each other / gave essentially the same results with the 2 g. substrate level as with 1 g. of cellulose. k2

Table 8 Study on the Additive Effect of Valeric Acid with Valine and Proline

Mg. of Mg. of Mg. of Grains of Valeric Acid Valine Proline Cellulose Digested

--- -- . 0.177

M rnt a w --- 0.585

10 --- 0.721

15 — --- 1.038

20 — --- 0.858 30 ------0.993 5 6 6 0.811 10 6 6 0.879 15 6 6 0.970 20 6 6 0.879 30 6 6 0.970

--- 6 6 0.7U3 Discussion

In the three natural materials studied (autolyzed yeast, casein hydrolysate and alfalfa extract) the cellu- lolytic factor activity was consistently found in the same cnromatograpnic fraction (top portion of the phenol chromatogram). Paper chromatographic analysis of these active fractions showed that they all contained the amino acids valine, proline, leucine and isoleucine in common. In this work, no quantitative data was obtained on the amino acid concentrations in the natural materials studied. The objective was to determine the quantity of natural material which would elicit a maximum cellulolytic response, and to use that quantity as a basic unit in the fraction­ ation procedures. The experiments presented in Table 3, on the separation of the active T.P.P.C. of yeast extract, indicate that the overall activity of the in vitro rumen fermentations was less than usual. In turn, this does not show a wide difference in cellulose digestion‘between the various components tested, but the results indicate that valine, proline and the leucines are cellulolytically active, while V-aminobutyric acid is Inactive. This Idea was further substantiated in the experiments utilizing the known amino acids. In the case of yeast extract and casein hydrolysate,

l|3 the stimulation of cellulose digestion by the prepared T.P.P.C. was approximately equal to the stimulation of the natural product. With alfalfa extract, the Dowex - 50 eluate and charcoal filtrate, as well as the T.P.P.C., exceeded the cellulolytic activity of the crude alfalfa extract. Prom the results of these experiments it is proposed that the cellulolytic factor activity of yeast extract, casein hydrolysate, and alfalfa extract is due to their content of the amino acids valine, proline, leucine and isoleucine. Any material which contains these free amino acids or is an in vivo ruminal source of them, would thus be a potential cellulolytically active agent assuming no inhibitors were present. Inhibition of cellulose digestion by higher levels of casein hydrolysate (from 100-150 mg. upward) was ob­ served In the studies with acid hydrolyzed casein. In­ hibition was found to be even more pronounced when enzy­ matically hydrolyzed casein was used as a factor source. Hershberger (32) has also reported this observation. An inhibition of cellulose digestion can be observed with an excess of known amino acids, and has even been observed with higher concentrations of the cellulolytically active amino acids. Hershberger ejfc aL (33) have reported the inhibition of cellulose digestion by the amino acid alanine. Hall et al. (29) have reported that partial hydrolyaates (acid, alkali and enzymatic) of casein are cellulolytically active, while complete hydrolysis re­ sulted in the loss of this activity. This suggests that their loss of activity upon complete hydrolysis may be caused by an excess of liberated amino acid3. The partial hydrolysates were possibly found to be active because of the valine, proline and the leucines being released from casein, and the total concentration of the liberated amino acids was not yet at the inhibitory level. The results in Figure 5, on the cellulolytic factor activity of alfalfa extract and the fractions propared by Dowex - 50 and charcoal treatment, show that the lower levels of crude alfalfa extract gave the maximum cellulose digestion obtainable from this material. Addition at higher levels resulted in a decrease in cellulolytic activity. When the crude alfalfa extract was fractionated with Dowex - 50 ion exchange resin, the Dowex - 50 eluate possessed approximately the same activity at the lower levels, but as the concentration of this material was increased, cellu­ lose digestion also increased. This suggested the possibil­ ity of an inhibiting substance present in the crude alfalfa extract, which was roraoved by the Dowex treatment. This inhibition observed in the crude alfalfa extract was pre­ sumably not caused by an excess of amino acids, because the amino acid concentration in the alfalfa extract and the ^ 6 Dowex - 50 eluate should have been about equal. The results obtained with the substrate level studies indicated that a problem of limiting substrate concentration was being encountered with one gram of cellulose. Thus, the rate of cellulose digestion would be less than the maximum rate; the velocity of an enzyme catalyzed reaction being dependent in certain cases upon substrate concentra­ tion (26). When the substrate concentration is limiting (all of the active sites of the enzyme are not occupied), the reaction is of the first order and the velocity is dependent upon the substrate concentration. When the level of substrate is increased to the point that all of the enzyme sites are occupied, then the reaction proceeds at its maximum rate, and the reaction is of zero order. Using one gram of cellulose substrate, about 0.75 g- was the maximum amount of cellulose digested, however, with two grams of cellulose substrate, digestion of as much as 1.0 to 1.3 g. of cellulose has been observed in the same time period. Additive effects of the various cellulolytic factors was then studied with two gram substrate level. No appreci­ able additive effects were observed that had not been pre­ viously observed at the one gram level, though the actual amount of cellulose digested was proportionally increased. The only additive effects observed in this study were with k7 valine and proline or the leucines and proline. Valine and the leucines appeared to function in somexfhat the same manner, since they both exhibited an additive effect with proline but not between themselves. Since the completion of this work, an article by MacLeod and Murray (39) has appeared in the literature, reporting that the amino acids valine, leucine and isoleucine stimu­ late in vitro cellulose digestion by washed rumen bacteria. MacLeod and Brumwell (38) had previously reported that a mixture of 18 amino acids stimulated cellulose digestion by washed rumen micro-organisms in vitro. In the latest report, examination of the amino acid mixture revealed that valine, leucine and isoleucine accounted for most of the stimulation observed. In their discussion, they mention­ ed that in general the complete mixture gave at least the same and sometimes slightly more activity than the above mentioned amino acids. In the light of the work reported here, this Increase in cellulose digestion may be caused by the additive effect of proline with valine, leucine and isoleucine. The possible mechanisms involved In the metabolism of valine, proline and the leucines in the in vitro rumen fermentation will be presented in Part II of this disserta­ tion. Summary

The cellulolytic factor activity of autolyzed yeast, casein hydrolysate and alfalfa extract was isolated by Dowex - $0 ion exchange resin treatment, charcoal (Darco G-60) treatment and large scale paper chromatography. The assay method used for this cellulolytic activity was the in vitro rumen fermentation technique. Factor activ­ ity of the materials studied was found to be in the top portion (above R^. 0.65) of the large scale phenol chromato­ grams. Further fractionation of the eluate from this section (using yeast extract as the starting material), by paper chromatography, indicated that the cellulolytically active factors were valine, proline, leucine and isoleucine. Water extracts of this portion, from all three materials, contained, these amino acids in common. The known amino acids valine, proline, and the leucines were found to be cellulolytically active when used in the in vitro rumen fermentation. Proline exerted an additive effect in combination with either valine or the leucines, while valine arid the leucines when tested together did not show this effect. A study was made on substrate level in the in vitro rumen fermentation, which indicated that the problem of limiting substrate concentration is encountered using one gram of cellulose. In the remainder of the experiments,

1*8 U9 two grama of cellulose substrate were utilized. Valeric acid (one of the cellulolytic factors of rumen juice), the amino acids (valine, proline, and the leucines), and yeast extract did not show any appreciable additive effects upon cellulose digestion when tested in various combinations. Part II

Studies on the Metabolism of the Isolated Growth Factors (Valine, Proline, LeucineT and Isoleucine)

Literature Review The identification of the amino acids (valine, proline, leucine, and isoleucine) as the components from autolyzed yeast, casein hydrolysate and alfalfa extract which in­ creased cellulose digestion in vitro, suggested that a study of their metabolism, directed toward the illucida- tion of their function In this process, would be of extreme interest. The amount of Information available on the metabolism of these compounds by anaerobic micro­ organisms Is rather limited, but several reports have appeared which indicated promising methods of approaching the problem. In 1952, El-Shazly (2l+), working with whole rumen fluid and washed suspensions, reported that the main reaction products obtained from the incubation of rumen bacteria with casein hydrolysate were ammonia, carbon dioxide and volatile fatty acids. Analysis of the volatile fatty acids, by gas-liquid partition chromatography, revealed

that straight and branched chain C2 to C£ fatty acids were present. When the concentrations of the fatty acids pro­ duced by incubation with casein hydrolysate were compared 50 51 with the concentrations normally present in rumen fluid (23), an increase in the branched chain and fatty acids was observed. Paper chromatographic analysis of the reaction mixtures indicated a general decrease in the concentration of all the amino acids, with a complete disappearance of phenylalanine, tyrosine and proline. A new spot consistently appeared on the chromatograms and was subsequently identified as 5-aminovaleric acid. On incubation of rumen liquor with proline alone, there was no obvious disappearance of proline, nor was any & -amino­ valeric acid formed. But, upon incubation of proline and alanine with rumen liquor, both decreased markedly in con­ centration and 8-aminovaleric acid was produced. Alanine, incubated alone with rumen liquor, simply showed a decrease in concentration. On the basis of these results, El-Shazly (2lj.) concludes that £ -aminovaleric acid is formed by a Stickland reaction between alanine and proline. Stickland (50), working with washed suspensions of Clostridium sporogenes, had observed that they were capable of activating certain amino acids as hydrogen donators (esp. alanine, valine and leucine) and other amino acids as hydrogen acceptors (glycine, proline and hydroxyproline). In a later report, Stickland (5l) found that these suspen­ sions reduced proline, at the expense of the oxidation of alanine, to give 8-aminovaleric acid. Neuberg (1911) and 52 Ackerman (1911) (as cited by Stickland) (5l) reported that incubation of cultures of mixed putrefactive bacteria with proline gave rise to S -aminovaleric acid. Niaman e_t al. (lj.3) have reported that a number of Clostridia, species and several other anaerobic micro-orgamisms are capable of carrying out a Stickland reaction between alanine and proline. In general, they found that only the proteolytic anaerobes could catalyze this type reaction; the non- proteolytic anaerobes and facultative anaerobes were in­ capable of carrying out this reaction. On the basis of his observations, El-Shazly (2lp) postulated that the amino acids valine, leucine and iso­ leucine act as hydrogen donors, while proline serves as a hydrogen acceptor in the fermentations with casein hydro­ lysate. Thus, valine, leucine and isoleuclne would be oxidized to the branched chain and C^ volatile fatty

acids, and proline reduced to 6 -aminovaleric acid. He further points out that other reducible substances may function in the place of proline if carbohydrate is being fermented simultaneously. Cohen-Bazire et «Q. (19) have reported that valine, leucine and isoleucine are oxidatively decarboxylated and deaminated by Clostridium sporogenes in the presence of proline, by means of the Stickland reaction. Isobutyric, Isovaleric and optically active valeric acids are the end 53 products formed. In studies with Escherichia coli, on valine, leucine and isoleucine biosynthesis and metabolism, the corres­ ponding ot-keto acids have been found to be the Immediate precursors or first metabolic product of these amino acids (l;i|6;5^-j55) • A transaminase has been isolated from E. coli which produces glutamic acid from e*-ketoglutaric acid. Valine and leucine are excellent amino donors In this re­ action, and isoleucine Is active to a lesser degree (25)• A similar pathway apparently exists in Torulopsia utilis (52). Proline Is apparently metabolized in E. coli by the same path as in mammalian tissue, with the end product being glutamic acid which can then enter the citric acid cycle (26). Roberts (lip) has reported that acetone powders of E. coli can catalyze the transamination of ^-aminovaleric acid with<*-ketoglutaric acid.

Kinnory et al. (3 6 ), working with intact rats and rat liver homogenates, studied valine metabolism using DL-Valine- lp, Their results Indicated that valine was oxidative­ ly deaminated to -ketoisovaleric acid, and then decarboxy- lated to isobutyric acid. Isobutyric acid is then converted through an intermediate step to propionic acid. Similarly, leucine and isoleucine are believed to be metabolized through the «A-keto acid and subsequent decarboxylation stages (2 6 ). Since Eentley ejb al. (3;5) and Bryant and Doetsch (9) have 3hown the volatile fatty acids (isobutyric, valeric,

isovaleric, 2 -methylbutyric and caproic acid) to be growth factors for rumen organisms, the concept that the amino acids were active because of their conversion to the corresponding fatty acids was very inviting. All of the known major metabolic pathways for valine, leucine and Isoleucine appear to involve these acids, and thus one might expect them to be intermediates or end products in the metabolism of these amino acids in the in vitro rumen fermentation. Experimental Procedures

Amino Acid Analysis Analysis of the in vitro rumen fermentation mixtures for free amino acids requlrod purification and a rather large degree of concentration. At the end of the fermenta­ tion period, 5 ml. of 50% trichloroacetic acid was added to the reaction fla3k, and the flask volume brought to 100 ml. with distilled water. Two, twenty-five ml.

aliquots were taken, placed in 5 0 ml. lusterold test tubes, and centrifuged for 1 hi’, at 1600 rpm. Each TCA supernatant was decanted into a second 50 ml. lusteroid tube, and the TCA sediment discarded. Dowex - f>0 ion exchange resin, in the hydrogen cycle, was then added to each tube, and the mixture allowed to stand at room tempera­ ture for about a half hour, .with intermittent mixing. The mixture was next centrifuged, and the supernatant discarded. Impurities not adsorbed on the resin were removed by re- suspending the material in distilled water and repeating the centrifugation and decantation steps. The amino acids were eluted from the resin by adding approximately 25 ml. of 50% Nll^Oii to each tube. The contents were stirred inter­ mittently over a two hour period, after which the Dowex - 50 was allowed to settle, and the 50% Nhj^Oh supernatant was decanted through a filter paper. The resin was transferred to the filter and washed with additional 50% Nh^Oh to insure

5 5 56 complete elution. The filtrates from both tube3 were combined, to give a final volume of between 100 and 200 ml. This eluate was concentrated to approximately 2 ml. by boiling on a hot plate, which also served to remove the ammonia. Thus, a degree of purification plus a twenty-five fold concentration was achieved. The amino acid composition of these preparations was investigated using one and two dimensional paper chromato­ graphy. The solvents utilized were the same as those mentioned in the experimental section of Part 1.

In Vitro Rumen Fermentation Technique Using Valine-1-C^- The in vitro fermentation technique used for the work with valine-l-C1-^ was identical to that described in Part I, except for several changes in the physical apparatus.

The CC>2 emerging from the reaction flask was passed through

CC>2 scrubbers filled with 6N NaOii, so that the GO^ was trapped as A trap was placed between the manifold and the reaction flask to prevent the radioactive contents from siphoning back into the manifold because of any

sudden drop in CO2 pressure. The entire apparatus was set up in a hood, to minimize possible contamination of the laboratory area. In all of the studies reported in this part of the dissertation, the two gram cellulose sub­ strate level was utilized. 57 Determination of m-Ketoisovaleric Acid The**~keto acids in the in vitro rumen fermentation mixtures were determined by conversion to their 2,1+- dinitrophenylhydrazine (DNPh) derivatives, and subsequent identification by paper chromatography. There are a number of procedures in the literature for the determination of

*-keto acids by this means (1 7 ;22;37;k%)> and the procedure utilized in this study is a combination of these techniques plus several modifications. At the end of the in vitro fermentation period the reaction mixture was deproteinized by the addition of TCA and subsequent centrifugation, since the presence of protein causes the formation of emulsions in the extraction of the DllPh derivatives. Five ml. of $0% TCA were added to the reaction mixture, and the total volume brought to 100 ml. with distilled water. Two 25 ml. aliquots were taken, placed in 50 ml. lusteroid tubes, centrifuged for one hour at 1600 rpm, and the TCA insoluble sediment was discarded. The TCA supernatant was treated with Dowex - 50 ion ex­ change resin (in the hydrogen cycle) by adding the resin to the supernatant in 5 0 ml. lusteroid test tubes, mixing the contents, and then centrifuging the resin out of suspension. The supernatants from the Dowex - 50 treatment were pooled, and 2 ml. of 0.5% 2,U-fli^itrophenylhydrazine in 6N HC1 were added. This mixture was allowed to stand at 58 room temperature for 30 minutes. At the end of this time, the mixture wa3 transferred to a separatory funnel, and the DITPn derivatives were extracted from the aqueous

solution with three successive 1 5 ml. portions of diethyl ether. The ether layers were combined and extracted with 15 ml. of IN Na^CO^, and the solvent layer discarded. The

Na2 C03 solution, containing the DNPN derivatives of keto acids, was washed with an additional 10 ml. of ether. The carbonate layer was cooled immediately (0-lj.oC), acidified with 5 ml. of 6N NCI, and the DNPN derivatives re-extracted with three successive portions of ether - 10,5 and 5 ml. respectively. The three extracts were pooled, dried by filtration through anhydrous Na2S0^, and- the ether was evaporated at room temperature under a gentle blast of air. The dry DNPN derivatives were then dissolved in the smallest possible volume of absolute ethanol. The DNPN derivative of ©(-ketoisovaleric acid was prepared by adding an excess of 0.5% 2 , dinit rophenyl- hydrazine in 6N NCI to a water solution of the acid. After standing for a short time at room temperature, the DNPN derivative of «*-ketoisovaleric acid precipitates from solution. The precipitate was filtered out, washed on the filter paper with distilled water and dried. The crystals of <*»-ketoisovaleric acid phenylhydrasone thus obtained, were then dissolved in a small volume of absolute ethanol. 59 Chromatographic investigation of this material (as describ­ ed below) revealed that traces of impurities were present, but the Rjr. value of the major component corresponded with the known Rp value for ot-ketoisovaleric acid phenylhydrazone in the butanol:ethanol:0,5N Nh^Oh solvent system (31). For chromatography, the DNPh derivatives were applied to the paper in the alcohol solvent as one inch streaks. Application in absolute alcohol is advantageous because the solvent evaporates rapidly, allowing the re-application of further material. Streaks are used in place of spots, because this allows the application of larger amounts of material to the paper and did not impair the separation. The solvent system described by El Hawary (22), butanol: ethanol: 0.5N NH^OH (70:10:20), gave a very satisfactory separation of the DNPh derivatives. Since a number of the other solvents described in the literature gave unsatis­ factory results, a second solvent system consisting of

tertiary amyl alcohol:pyridine:water (7 0 :2 0 :1 0 ) was developed and utilized. After development, the chromatograms were dried at room temperature in the hood. The DNPh derivatives of ■vketo acids are yellow in color, and thus the separated constituents were visible without further treatment. The radioactivity of the constituents was counted by elution and plancheting methods along with a gas-flow chromato- graphic scanner (Model C-100 Actigraph; Nuclear Instru­ ments). The chromatographic scanning technique is not a sensitive as the eluting and pl&ncheting method, and therefore this latter method was utilized for those samples which were low in radioactivity. Results

Investigation of Added Amino Acids The various amino acids were added to the basal medium of the in vitro rumen fermentation, alone and in combina­ tion, and the subsequent reaction mixtures were analyzed for amino acid content. In the first experiment, reported in Table 9, several flasks were analyzed at thirteen hours and the remaining flasks at thirty hours for comparison purposes. The amino acid analyses were carried out by paper chromatographic methods as described in the experi­ mental procedures. Minute traces of several amino acids were found in the basal flask after the thirty hour fermenta­ tion period, and thus the detection of traces of these same amino acids in the fermentation mixtures under study was considered normal, and probably not directly concerned with the reactions of the added amino acids. 1'n other words, the amino acid pattern of the basal flask was subtracted from the pattern of the flasks with added amino acids. Thus, the data presented in Table 9 shows only the changes observ­ ed in the amino acids added to the basal medium. Prom the results shown in Table 9 it can be seen that the concentra­ tion of valine decreases as the length of the fermentation increases, though approximately half of the valine remains at the end of the fermentation period. Proline concentra­ tion also decreased with time, and the proline had completely 61 Table 9 Amino Acid Composition of Fermentation Mixtures

Amino Acidsa Addition to Time Basal Medium (hours) Valine Proline 6-aminovaleric acid

None 30 * * M M ------O '1' X , j DL-Valineb ------

DL-Valine plus L-Prolinec 30 ------

DL-Valine plus L-Prolinec 13 -i'riHKS- ** ~t7~4\

< > < t L-Leucine plus L-Prolined 13 (W*

a. A visual quantitative evaluation of the q u a n t i t y of amino acids present was made, ranging from # (trace) to *:k h k :- (large quantity). b. 20 mg. of DL-Valine per 100 ml. c. 32 mg. of DL-Valine plus 16 mg. of L-Proline per 100 ml. d. 16 mg. of L-Leucine plus 16 mg. of L-Proline per 100 ml. disappeared at the end of thirty hours. The appearance of 6-aminovaleric acid was noted in those flasks analyzed at thirteen hours, with a subsequent decrease in concentra­ tion by thirty hours. In the identification of 6-amino- valeric acid, it was noted that the new spot corresponded in value to those reported for 6-aminovaieric acid in aqueous phenol (7) and butanol:acetic acid:water (53). Addition of known 6-aminovaieric acid to the unknown did not result in the appearance of any new spots when tested in several solvent systems. This was true using both one- and two-dimensional paper chromatography. Analysis of the flask to which L-leucine and L-proline had been added, revealed that this amino acid had completely disappeared after thirteen hours. It had been suspected previously that D-valine was not utilized by rumen bacteria and the concentrations of L-valine desired for the experi­ ments in this study were obtained by doubling the amount of DL-valine. Since approximately half of the added DL- valine appeared to be present at the end of thirty hours, in contrast to the complete disappearance of L-leucine in thirteen hours, this supposition would appear to be correct In order to determine if D-valine is utilized by.rumen bacteria, samples of D-valine and L-valine were obtained and tested for cellulolytic factor activity in the in vitro rumen fermentation. The results of this study are pre- 6I{. sented in Table 10. From the data obtained, it appears that on an equal weight basis, DL-valine is approximately half as active as L-valine, while D-valine is almost com­ pletely inactive. This comparison holds true whether the valines are tested alone or in combination with L-proline. A second experiment on the fate of the added amino acids was run, using L-valine. The results of this second experiment are shovm in Table 11. In this experiment, valine and proline were again observed to decrease in con­ centration as the fermentation proceeded. The appearance of &-aminovaleric acid was again noted in the thirteen hour samples, with a decrease in concentration occurring by hours. Since 6-aminovaleric acid appeared when proline was added alone to the basal medium, one can conclude that this amino acid is formed by ring cleavage and reduction of proline. That 6-aminovaleric acid arises from proline would be in agreement with the results of El-Shazly (12), except in this work the presence of a hydrogen donating amino acid was not required. Figure 10 shows a diagrammatic separation of the amino acids from flasks with added valine and proline. The glutamic acid spot was also observed in the basal flask, and thus is probably not directly concerned with the re­ actions of the added amino acids. In summary, the amino acids valine and leucine were 65

T a b l e 1 0

Cellulolytic Factor Activity of D-Valine, DL-Valine and L-Valine Alone and in Combination With Proline

Addition to Grams of Cellulose Digested Basal Medium 3/15/57 3/21/57 3/25/57 Average

None .202 .095 .068 .122 D-Valinea .296 .008 .122 .li|2

DL-Valine .376 .282 .283 .3 1 I4. L-Valine . I4.I6 .^83 • 523 L-Proline . I4.I6 .537 .2 0 2 .385 D-Valine plus L-Proline .2 8 2 .350 .577 .14-03 DL-Valine plus L-Proline .657 .76i|. . 8t|l|. .755 CD H L-Valine plus L-Proline -0 1 .1 1 2 .939 .956

a. A concentration of 6 mg. per 1 0 0 ml . was used when the amino acids were tested alone, and 6 mg. per 1 0 0 ml. of each amino acid when tested together Table 11

Amino Acid Composition of Fermentation Mixtures

Addition to Time Amino Acidsa Basal Medium (hours) Valine Proline 6-aminovaleric acid

L-Proline^ 13 ------

L-Valine plus L-Prolinec 13 \r

u « > L-Valine plus L-Prolinec 2lf " I*” "li*

a. A visual quantitative evaluation of the quantity of amino acids present was made, ranging from -* (trace) to *:k h k ;- (large quantity). b. 10 mg. of L-Proline per 100 ml. c. 10 mg. each of L-Valine and L-Proline 67 t

VoWae u 0 o Pro fine 0 0 0 b-HHg- Voleric acid 0 o 0 0 O CM X Glutomic ID .__ o ° o ,<= If} 0 „ CM «0

go r o »*• p v+p v + p p 13hr*. 24 hr*. 30hr«. I3hr«.

Figure 10. Diagrammatic separation of the amino acids from flasks with added valine and proline (V and P), determined at 13, 2h and 30 hours, and of added proline. (P), determined at 13 hours 68 observed to decrease in concentration as the fermentation proceeded. This would be in agreement with the hypothesis that these amino acids are cellulolytically active be­ cause of their conversion to the corresponding branched chain C^ and C^ fatty acids. Proline was converted to

6-aminovaleric acid, which was used up as the fermentation progressed.

Rumen Fermentation (In Vitro) with Valine-l-C-1-^

When DL-valine-l-C^- was added to the in vitro rumen fermentation medium, a .large portion of the radioactivity was recovered as c1^o2 , This can be seen readily from the data presented in Table 12. In the first experiment reported in Table 12, the inoculum was autoclaved before being added to the flask. Since only a very small number of counts were recovered as in this case (9%), it can be concluded that carboxyl exchange of valine-l-C^, with the GO^ used to maintain anaerobic conditions, does not account for the large proportion of C ^ O which is found. Thus it appears, that certain of the rumen micro­ organisms are capable of decarboxylating valine. In some cases, particularly the last experiment In Table 12, the recovery of radioactivity was quite low. But even then, the amount of radioactivity recovered as C-^0 is signifi­ cantly higher than that observed in the first experiment, and this substantiates the conclusion that valine undergoes Table 12

Decarboxylation of DL-Valine-l-C-4 by Rumen Micro-organisms In Vitro

Radioactivity- Radioactivity Additions to Grams Recovered as c4-0p Remaining in Flask % Recovery Cellulose % of Added % of Added of Total Basal Medium0 Digested Count s/min* Counts Count s/min. Counts Added Counts

DL-Val I no-1 - C^!-b .000 100,000 9.0 1,770,000 88.6 97.6

DL-Valino-l-c4 430 730,000 36.5 7iH429 37.0 73.6

DL-Valine-l-C1^ .523 1 2 ,6 8 0 ,0 0 0 U3.1 16,300,000 5 5 4 96.6 DL-Valine-l-c4c .000 8,168,750 2 6 4 13,781,250 14* 6 71.1

DL-Valine-l-G1^ 0 .000 8,168,750 2 6 4 1 3 4 0 0 , 0 0 0 [{-34 69.8 DL-Valine-l-c4 .939 715,000 35.8 5 5 0 ,0 0 0 27.5 63.3

DL-Valine~l~c4 .885 Ij.16,880 20.9 Lj.0 5 ,0 0 0 20.3 4L.1

a. In these experiments, the basal medium included 6 mg. of L-proline per 100 ml., and enough DL-or L-valine to give a final concentration, in combination with the added DL-valine-l-c4, of 6 mg. of L-valine per 100 ml. b. Control, inoculated with autoclaved.cells. c. In this experiment, both flasks were connected to a common C0p trap. The counts rooovered as c402 in each case aro thus half of the total counts found as 70 decarboxylation.

It was shown in the previous section that D-valine is not active as a cellulolytic factor, and appears to be rather inert throughout the fermentation. One would therefore expect that approximately half of the total counts added as DL-valine-l-C^ should be present in the reaction flask at the end of the fermentation period.

The data in Table 12 would appear to support this idea, and In no case was more than half of the total radioactiv­ ity recovered as C^-C^.

When the reaction mixtures, obtained upon incubation with DL-valine-l-C^-, were precipitated with TCA and centrifuged, almost all of the radioactivity was recovered in the TCA supernatant. Fractionation of the TCA super­ natant or the whole reaction mixture by treatment with

Dowex - j?0 ion exchange resin (hydrogen cycle), resulted in approximately 9$% adsorption of the radioactivity on the resin. The radioactivity could be eluted from the resin with $0% Nh^Oh; and the resulting eluate was con­ centrated and subjected to paper chromatography. Radio­ autographs (using X-ray film) were made of the resulting chromatograms, and except for a trace of radioactivity at the origin, all of the radioactivity was found to be present in valine. This would then be rather strong evidence that the major portion of the radioactivity 71 remaining in'the reaction mixture after the fermentation period was D-valine-l-cA^. The only other amino acids present in this Dowex - 50 eluate were proline, S-amino- valeric acid, and a trace of glutamic acid. Analysis of the radioactivity not adsorbed by Dowex - 50 ion exchange resin will be described in the next section.

The bacterial cells (TCA sediment) were subjected to hydrolysis In 6N hCl, and the hydrolysate waG analyzed by paper chromatography. The resulting chromatogram was divided into one inch segments which were eluted with hot water, and the radioactivity of the eluates determined by counting in a gas-flow counter. This technique is much more sensitive than the radioautograph technique, and was employed because of the.low number of counts in the bacter ial cells. When the radioactivity data were compared with ninhydrin developed chromatograms run simultaneously, tne majority of the activity present corresponded with the valine spot. A trace of radioactivity was noted near the solvent front, but no amino acids were present in this zor and the Identity of this source of radioactivity is un­ known. In view of these results, and the decarboxylation data, it is felt that valine does not function as an essential amino acid required by the rumen bactei’ia; where by the amino acid is incorporated directly Into the bacter ial protein. 72

In summary, DL-valine-l-C1^- was shoxm to be decar- boxylated in the jLn vitro rumen fermentation. The majority of the radioactivity remaining in the reaction mixture was found to be in valine, presumably D-valine-l-C-^-.

Isolation of Radioactive *-Ketoi3oyaleric Acid

In view of the data presented in the two previous sections, it appeared extremely likely that the cellulo- lytic factor activity of valine was the result of its conversion to isobutyric acid. If this were true, one would expect the reaction to occur in two steps; the first being oxidative deamination, followed by decarboxylation.

Since the decarboxylation reaction would remove the radio­ active tag from the valine used in these studies, an attempt wa3 made to isolate radioactive <*-ketoisovaleric acid, which would be the logical intermediate.

Several attempts were made to develop a column chromato­ graphic method for the determination of «*-ketoisovaleric acid; silica gel was used as the adsorbent, and mixtures of benzene and butanol as the eluting solvents. It was not possible to obtain a clean separation by this method, even when a known sample of o^-ketolsovalerlc acid was added to the column alone. Thus, the column method was abandoned in favor of paper chromatography.

The conversion of <*-keto acids to their 2,I|.-dInitro- phenylhydrazine (DNPh) derivatives, and subsequent paper chromatographic separation of these derivatives is a

common technique today, and thus this method was adopted.

The details of this method are given in the section on

experimental procedures. Since

adsorbed by Dowex - $0 ion exchange resin (in the hydrogen

cycle), the Dowex- 50 filtrates obtained from the primary

fractionation of the reaction mixtures for amino acid

analysis, reported in the previous section, were analysed

for radioactive o<-ketoisovaleric acid. The Dowex - j?0

filtrate was seeded with known ot-ketoisovaleric acid, which would act as a carrier in the isolation procedure.

The Dowex - 50 filtrate was then carried through the pro­

cedure for the conversion of <*-keto acids to their DNPh derivatives, and the resulting DNPh derivatives were

separated by paper chromatography. Three distinct yellow

zones were visible on the resulting chromatograms, one wa3 very large and deep yellow in color, while the other two were present only in trace amounts. When known

evketoisovaleric acid was converted to its DNPfi derivative and chromatographed, the same three spots were found. This would then indicate that the known ^-ketoisovaleric acid sample was impure, and the appearance of these small spots in the DNPJd derivatives from the reaction mixture is a result of the seeding technique. The Rp value of the heavy spot obtained from the known sample of

ponded with the known value for e*-ketoisovaleric acid

phenylhydrazone (3 1 ). V.Then the -ketoisovaleric acid phenylhydrazone spot obtained by chromatography of the DITPn derivatives of the Dowex - 50 filtrate was cut out, eluted with IN NaOil and counted in a gas-flow counter, only a few counts per minute above background were obtain­ ed. It was felt that contamination could easily account for this small number of radioactive counts, and thus no conclusions could be made. Repetition of this determination with the Dowex - 50 filtrate from several flasks gave similar results. In all of the fermentation mixtures tested,< a total of 12 mg. of DL-valine per flask had been used. An experiment was then set up, in which 32 mg. of non­ radioactive DL-valine was added, and an attempt was made to isolate < -ketoisovaleric acid from this mixture. Paper chromatograms of the DNPh derivatives obtained revealed trace amounts of the same three spots which had been ob­ served previously. Thus, it can be concluded that e*-keto- isovaleric acid is formed to a slight degree upon the incubation of valine in the in vitro fermentation. The appearance of the other two spots Indicates that these compounds are also normally present in the mixture, and are not necessarily introduced by the seeding technique 7b employed in the radioactive experiments. At this point, it was thought that possibly the reason

for the lack of success in isolating radioactive c<-keto- isovaleric acid was the velocity at which the decarboxy­

lation reaction occurred. In other words, as soon as valine was oxidatively deaminated it might then be rapidly decarboxylated, with no accumulation of theo(-keto acid as an intermediate product. If by some means, the rate of this decarboxylation reaction could be slowed down, the intermediate might then accumulate to a slight degree and therefore be isolated. Since one of the most common methods for decreasing the rate of an enzyme catalyzed reaction is to lower the temperature, an experiment was set ^^p with DL- valine-l-C-1-^- in which the temperature of the in vitro rumen fermentation was maintained at 25°C. The fermentation was allowed to proceed for 20 hours, at the end of which time the e<-keto acid fraction, after being seeded with known ^-ketoisovaleric acid, was isolated by the DNPh procedure. The phenylhydrazone fraction thus obtained, was found to contain about 0 .2% of the total radioactivity added to the flask. In actual numbers, this amounted to approximately h0,000 counts per minute. This phenylhydrazone fraction was separated by paper chromatography, and three yellow zones were again visible. The -ketoisovaleric acid phenylhydrazone spot was very heavy, while the other two 76 components wore present only in trace amounts. When the •{-ketoisovaleric acid phenylhydrazone spot was cut out, eluted and counted for radioactivity, it was found to be quite active and accounted for almost all the counts pre­ sent in the phenylhydrazone fraction. These chromatograms were also counted in the gas-flow chromatographic scanner, which was connected to an automatic recorder. A tracing of one of these chromatograms and the corresponding graph of its radioactivity, obtained from the scanner, are shown in Figure 11. The area under the radioactive peak obtained from the DNPn derivative of ^-ketoisovaleric acid represents approximately 1200 counts per minute. The -ketoisovaleric acid should be labeled in the carboxyl carbon, but no investigations of this point were made. Since the actual number of counts per minute recovered as radioactive ®<-ketoisovaleric acid was so small in com­ parison to the total number of counts per minute added to the fermentation medium, the question arose as to whether or not a small amount of radioactive valine might not be carried over in the isolation procedure. When the Rf values for valine and the DlTPn derivative of tx-ketoisovaleric acid were compared in the butanol:ethanol:0 .5 N Nh^Oh solvent system, valine had an value of about 0 .2 5 * while o(-ketoisovaleric acid phenylhydrazone had an Rp of about 0.?5« Therefore, it can be concluded that the radioactivity 77

[ 0 0 OrIgln «-tl£79 Bovtnt ------> uov.iitmc .. Krom BuOH'EtOH'O.SN NH4OH (70:|0>Z 0)

Figure 11. Tracings of the chromatographic separation of the 2 ,lj.-dinitrophenyXhydrazine derivatives obtained from a fermentation with DL- valine-l-Cl^-, and the corresponding graph of its radioactivity as measured with a gas-flow chromato­ graphic scanner 78 found in the DNFn derivative of -ketoisovaleric acid was not the result of contamination from valine-l-ClU.

One further experiment wa 3 carried out, in which DL- valine-l-C-^ was incubated in the in vitro rumen fermenta­ tion for six hours at 39°C. It was believed that this shorter time period might allow the isolation of radio­ active ot-ketoisovaleric acid. The reaction mixture was treated as described previously, and approximately 0 .03% of the added counts were recovered in the phenylhydrazone fraction (approximately 8000 counts per minute). Upon subsequent chromatography, a small amount of radioactivity was found in the DNPn derivative of ©<-ketoisovaleric acid by- the elution technique. The gas-flow chromatographic scanner was not sensitive enough to pick up this small amount of activity.

Comparison of the values of the other two DNPH derivative spots, which were found in trace amounts in the reaction mixtures, indicated that these compounds might be acetone and pyruvic or acetoacetic acid. No further attempt was made to identify these compounds.

In summary, radioactive o<-ketoisovaleric acid was isolated from in vitro rumen fermentation mixtures when DL- valine-1 - C1^ was used as a cellulolytic factor. The quantity of radioactive ot-ketoisovaleric acid in­ creased when the temperature of the fermentation was lox^erod 79 from 39°C to 2^°C.

Cellulolytic Factor Activity of Possible Intermediates in VaYine', Proline and Leucine Metabolism

The experimental data presented in the previous sections

supports the idea that valine is oxidatively deaminated and

subsequently decarboxylated by rumen bacteria in vitro. Thus valine, ©<-ketoisovaleric acid and isobutyric acid should possess cellulolytic factor activity alone, and in combina­ tion with proline. Since it had been previously learned, from column and paper chromatographic methods, that the known -ketoisovaleric acid contained impurities, a crude type of assay was attempted to determine the quantitative relationship between L-valine and the impure o<-ketoiso­ valeric acid. The results of these experiments are shown in Table 13* and it appeared that doubling the quantity of

«*-ketoisovaleric acid would give a better representation of its cellulolytic factor activity.

Prom the limited studies on L-proline,- it was con­ cluded that this amino acid underwent ring cleavage and reduction to form 6 -aminovaleric acid. Aside from the fact that 6 -aminovaleric acid disappears from the fermenta­ tion mixture, its metabolic fate is unknown at the present time.

The amino acids valine and proline, and their pro­ posed metabolic breakdown products, were tested alcne and 80

Table 13

Relationship of L-Valine and ^-Ketoisovaleric Acid in the In Vitro Rumen Fermentation

Additions to Grams Cellulose Digested Mg. of Each Amino Acid Added Basal Medium r______0 3.0 6.0 9.0 12.0 1^.0

Experiment I

None .122 L-Valine .336 .831 .965 L-Proline .610). .601+ .711 ^-Ketoisovaleric Acid .336 .256 . 61+1+ L-Valine plus L-Proline 1.085 l.llj.0 1.193 ot-Ketoisovaleric Acid plua L-Proline . m .577 .8U5

Experiment 11

None .202 L-Valine . .JLj.031+03 . .1+161+16 .577 .577 *hk3 L-Proline .1+16 ot-Ketoisovaleric Acid .0 .015 1 5 .095 .095 . . 11+9li+9 .3^-9 L-Valine plus L-Prolinea .737.7 3 7 .8.8 1 1 7 7 .871 .871 .8 3 1 .657 «*-Ketoisovaleric Acid plus L-Prolinea .296 .296 .577 .577 .590 .590 .561+ .76 I4.

a. 6 mg, of L-Proline were added at each level. in all combinations in the in vitro rumen fermentation

for cellulolytic factor activity. The results of five

experiments are shown in Table II4. As can be seen in the Table, cellulose digestion in flasks containing valine,

proline, isobutyric acid or S-aminovaleric acid are signifi­

cantly different from the negative control, while the

addition of c<->etoisovaleric acid shows no appreciable

difference. Valine, in combination with proline or S-amino\raleric acid, possesses more cellulolytic factor activity than valine, proline or 6-aminovaleric acid alone. The same conclusions apply to isobutyric acid in combina­

tion with proline or ^-aminovaleric acid. In general, the standard deviations obtained for these experiments was

rather high, and is probably caused by the variability between the different fermentations. The results obtained within each experiment were more consistent, and all showed

the same over-all trend. The difference between the means

of ^-ketoisovaleric acid plus proline or S-amlnovaleric acid and proline or &-aminovaleric acid alone did not appear to be significantly different when they were com­ pared with the standard deviations included. Thus a paired t test was run on proline alone and -ketoisovaleric acid plus proline, and they were found to be significantly different at the level. The paired t test removes the variability between separate experiments. A paired t test Table 4

Cellulolytic Factor Activity of Proposed Intermediates in the Metabolism of Valine and Proline

Additions to Grams Cellulose Digested b Basal Mediuma 12/13/56 12/21/56 3/21/57 3/28/57 142/57 XSX

None .055 .095 .095 .068 . 0i}.2 .071 + .022

L-Vallne (V) .523 433 4.83 .523 416 436 . OH4

c<-Kotoisovflloric Acid .49 .008 .014.8 .176 .068 .090 + .071 (*<-K-iso-V.A.} Isobutyric Acid (Iso-B.A.) • 43 4 1 6 .350 .256 .269 -347 . 034

Proline (P) .14-83 .336 .537 .202 .381 + .132

6-Aminovaleric Acid .216 .256 496 483 . 214-3 .339 + . 138 ( 6 -Nhg-V.A.) V plus P .670 ' .81-5 1.112 .510 1.019 .831 + . 2li.6

V plus S-M^-V.A. .750 ,68ij. .858 .697 .871 .772 'T .089

o(-K-iso-V.A. plus P 496 .510 .858 .671 416 .590 + .176

°c-K-iso~V,A. plus S-Nh2 -V,A. .310 • 550 • 8oLj. .631 4-96 .558 4" .181

Iso-B.A. plU3 P 1.019 -845 1.286 1.01+5 1.019 1.043 + .158

130-B.A. plu3 S-Nh2 "V.A. .817 .965 .912 .564 .671 .786 + .167 All materials, alone or in combination, added at 5*12 x 10-5 moles per 100 ml. except t*-ketoisovaleric acid which was double that quantity. b. Moan and standard deviation. 83 between S-aminovaleric acid and o<-ketoisovaleric acid plus £-aminovaleric acid revealed that they were significantly different at the 11 level. The experimental results shown in Table llj. would appear to substantiate the proposed metabolic pathways of valine and proline. An experiment, similar in design to the one shown in Table llj., was set up to test the intermediate products in the metabolism of leucine. The actual amount of experi­ mental data obtained upon leucine was very small, simply

that it disappears upon incubation in the in vitro rumen fermentation, but it is believed that this amino acid may follow the same general path as valine (oxidative deamina­ tion and decarboxylation). If this were true, then the

intermediates would be c^-ketoisocaproic acid and isovaleric acid. These intermediates, along with proline and 8-amino- valeric acid, were tested for cellulolytic factor activity, and the results are shown in Table 15* Leucine does not appear to be as active a cellulolytic factor as valine, and this is reflected in an over-all lowering of the amount of cellulose digestion. The standard deviations observed with these experiments were higher than those in the pre­ vious experiment, but the over-all trend supports the proposed metabolic pathway for leucine.

Some interesting results were obtained when a time study was made of valine, proline and leucine and their Table 15 Cellulolytic Factor Activity of Proposed inter­ mediates in the Metabolism of Leucine and Proline

Additions to Grams Cellulose Digested Basal Mediuma 12/ 2 1 /5 6 4/ 2 /5 7 4/4/57 x 3x CO -d O + 1 None .095 . 0J4.2 .000 .OI46 • L-Leucine(L) . Il96 .229 .1 0 8 .278 + .198

e*-Ketoisocaproic Acid .229 .031^ .000 .088 + .123 (ot-K-iso-C.A. ) Isovaleric Acid .577 .216 .01+1 .2 7 8 ± .273 (iso-V.A.) L-Proline (P) .336 . 3U-9 .095 .260 ± -lk3 S-Aminovaleric Acid .256 . 2l|3 .000 .166 + . 1I4J-1- (6 -NM2 -V.A.) L plus P .657 -349 . 561+ .523 + .153

L plus <5-Nh2-V.A. .523 .il-56 .314-9 .1&3 ± *°88 o<-K-iso-C.A. plus P .336 .2 1 5 • 2lf3 .265 + . 06 I4. 00 H + 1 ot-K-iso-C.A. .376 .021 .282 .226 • plus 6 -Nh2 ~V.A. Iso-V.A. plus P .751 .14-03 .617 .590 + .176

Iso-V.A. .737 .,2 82 .1483 .501 + .228 plus 6 -N1I2 -V.A*

a. All materials, alone or in combination, added at 5*12 X 10-5 moles per 100 ml. except -ketoisocaproic acid which was double that quantity.

b. Mean and standard deviation. proposed breakdown products. These results are 3hovm in Table 16, and they lond support to the proposed pathways.

Samples were taken at Lp, 8, 12, 16, 2l(-, and 30 hours, but no cellulose digestion was observed until 16 hours, v/hen a slight amount occurred in some fla3k3. A sharp increase in cellulose digestion wa3 observed between 16 and 30 hours, as can be seen from the Table. Because of the similarity of the action between these compounds, it seems possible that the amino acids (valine and leucine) are converted to their corresponding fatty acids, and proline to 6 -amino- valeric acid, during the first sixteen hours, thus account­ ing for their activity. This idea is substantiated by the studies on the disappearance of the added amino acids. Since valeric acid is the major cellulolytic factor of rumen juice, it was believed that the •=< -amino homologue of valeric acid, norvaline, should be cellulolytically active. However, subsequent studies with this compound did not substantiate this thought. When S-aminovaleric acid was found to possess cellulolytic factor activity, it suggested that there is a definite specificity concerned with this straight-chain five carbon amino acid compound. The straight-chain compound acts in an additive fashion with the branched-chain compounds (valine, leucine, iso­ butyric acid and isovaleric acid). Since the c^-amino compound was inactive and the 8-amino compound active, it 8 6

Table 16 Time Study of Valine and Leucine Plus Proline Versus isobutyric Acid and isovaleric Acid Plus 6 -Aminovaleric Acid

Additions to' Grams Cellulose Digested Basal Mediuma hours 16 24 30

None „ — .202

Valine plus Proline .000 . 698 1.058

Isobutyric Acid plus S-NN^-V.A.k .015 .684 1 .0 7 2

Leucine plus Proline .000 .470 .817

Isovaleric Acid plus S-Nt^-V.A. .000 .350 .898

a. All materials added at 5*12 X 10-5 moles per 100 ml. b. 6-aminovaleric acid (^-Nh^-V.A.) 87 seemed of interest to test a , S -diamlnovaleric acid, more commonly known as ornithine. The ability of ornithine to partially replace proline as a cellulolytic factor in combination with valine is shown in Table 17. The sicnifi cance of this observation will be discussed in the next section. Table 1?

The Cellulolytic Factor* Activity of Ornithine

Additions to Grams Cellulose Digested Basal Medium8- j III Ave.

None .202 .0 0 0 .0 0 0 .067

L-Proline .630 .1+96 . 336 .1+87 DL-Ornithine .229 .0 0 0 .0 0 0 .079

L-Valine .336 . 256 .2 0 2 .265

L-Proline plus DL-Ornithine --- .309 .175 .21+2

L-Proline plus L-Valine .8 1 7 .898 1 .0 5 8 .921+

DL-Ornithine plus L-Valine . SOlp .1+56 .617 . 626

a. 6 mg. of the L-form of each amino acid per 100 m l . Discussion

The proposed pathways for the metabolic breakdown of valine and proline arc shown in Figure 12. The experi­ mental evidence obtained with valine-l-C^I and the factor activity of the proposed intermediates strongly supports these pathways. Considerably less data was obtained on the metabolic breakdown of leucine, but the results suggest that leucine follows a pathway similar to valine. The intermediates in the breakdown of leucine would then be t^-ketoisocaproic acid and isovaleric acid. The corres­ ponding intermediates which might be proposed for the breakdown of isoleucine were not available, and thus could not be tested for factor activity in the in vitro rumen fermentation. Assuming a similar pathway of oxidative deamination followed by decarboxylation, the breakdown products of isoleucine would be 3-methyl-<*-ketovaleric acid and 2-methyl-butyric acid.

These reactions are similar to the Stickland reaction (l9,*Lt-3,*50;5l), whereby a hydrogen donating amino acid (valine, leucine and isoleucine) reduces a hydrogen accept­ ing amino acid (proline). However in this study, proline was converted to £-aminovaleric acid in the absence of a hydrogen donating amino acid. This observation is in con­ trast to the findings of El-Shasly (2if), and might suggest that the breakdown of these compounds in the In vitro rumen

89 9 0

PROPOSED PATHWAYS

MHg G C C-C-C-GDOH t C CI I C-COOH V a tin * \/ Proline i 0 1 C-C-C-ODOH & h2n -c-c-c -c-cooh a-KttalscvatlBric 6-NHg-Voleric

I' . C-C-COOH I 8 C liofrutyiriir

1 8

Figure 12. Proposed pathways in the metabolic breakdown of valine and proline 91

fermentation does not comply strictly with the Stickland reaction. In these studies on the cellulolytic factor activity of the amino acids, a two component system Is required for maximum activity. One component is branched-chain (valine, leucine, Isoleucine' and their corresponding fatty acids), while the other component appears to be straight- chained (proline, 6-aminovaleric acid and ornithine). Bryant and Doetsch (9) have reported that a rumen isolate, Bacteroides succinogenes, requires a combination of a branched-chain fatty acid (isobutyric, isovaleric or 2- methyl butyric acid) and a straight-chained acid (C^ to Cg) for growth in vitro. Their work then substantiates the two component requirement observed in the present study, and the probable conversion of the amino acids valine, leucine and isoleucine to their corresponding fatty acids (one carbon less in length). El-Shazly (2l±) has proposed that &-aminovaleric acid might be converted to valeric acid by rumen micro-organisms. If this were the case, then proline would contribute the straight chain component required in the two component system. Roberts

(i|5) has reported that acetone powders of E. coli catalyze the transamination of 6-aminovaleric acid with ok-keto- glutaric acid, thus converting it to glutaric semialdehyde. This compound was not available, but glutaric acid was 9 2

tested for factor activity and found to be inactive. It is then proposed, that the amino acids valine, leucine and isoleucine function as cellulolytic factors because of their conversion to fatty acids. Aside from this study, the cellulolytic factor activity of these fatty acids has been conclusively demonstrated by Bentley

et al. (3;5). Proline functions in an additive fashion with these compounds, but the actual breakdown products

beyond S-aminovaleric acid are unknown. Suggestions for further work on this problem would include the use of uniformly labeled ClU- amino acids. Thus, the radioactive tag would not be completely lost by de­ carboxylation, and the reaction mixture could be analyzed for radioactive fatty acids. Since these are Iso-acids,

a gas-liquid partition chromatographic apparatus would be an invaluable aid for these analyses. Radioactive proline would provide a means by which to study the breakdown of &-aminovaleric acid, to determine if a straight chain acid is formed. V/oods (57) has reported that washed suspensions of Clostridium sporogenes will reduce ornithine to & -amino- valeric acid (1|7?0 via a Stlckland type reaction. Stadtman (1|9), working with an amino acid-fermenting Clostridium, has observed that 6% of ornithine-2-ClU is reduced to £-aminovaleric acid, and 1% to proline. Presumably for 93 this reaction to occur, ornithine must undergo reductive deamination at the -position. hence the cellulolytic factor activity of ornithine, as a proline replacing agent, can possibly be explained through its conversion to 6-aminovaleric acid. Summary

Paper chromatographic analyses of fermentation mixtures,

to which valine, proline and leucine had been added alone and in different combinations, revealed that the amino

acids valine and leucine decrease in concentration as the

fermentation proceeds. Proline was found to be reduced to 5-aminovaleric acid, which also had decreased markedly

in concentration by the end of the fermentation period.

D-valine was found to be inactive as a cellulolytic factor

in the in vitro fermentation.

When DL-valine-l-C^ was added to the in vitro rumen

fermentation, a large portion of the radioactivity was recovered as Of the radioactivity remaining in the fermentation mixture, the majority appeared to be present as valine, presumably D-valine-l-C^-.

Analysis of the reaction mixtures obtained from the incubation of DL-valine-l-C^ij-, revealed that a small amount of radioactive o{-ketoisovaleric acid was present. An in­ crease in the quantity of this compound was noted when the temperature of the fermentation was decreased. The proposed breakdown products of valine ( -ketoiso- valeric acid and isobutyric acid), proline (£-aminovaleric acid) and leucine (c*-ketoisocaproic acid and isovaleric acid) were tested for their cellulolytic factor activity in the in vitro rumen fermentation. These compounds were capable

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51}-. Umbarger, h. S., and Magasanik, B. Isoleucine and Valine Metabolism of Escherichia coli II. The The Accumulation of Keto Acids. J. Biol. Chem., 189, 287 (195D. 55* Umbarger, h. E., and Magasanik, B. Isoleucine and Valine Metabolism of Escherichia coli IV. Competitive Interaction in the T’ransamination Reactions of Isoleucine and Valine. J. Am. Chem. Soc., 7k, U256 (1952). 56. Wasserman, R. h., Seeley, H. W., and Loosli, J. K. The Physiology and Nutrition of a Rumen Lacto­ bacillus. J. Animal Sci., 12, 935 (1953). 57* Woods, D. D. Studies in the Metabolism of the Strict Anaerobes (Genus Clostridium) V. Further Experi­ ments on the Coupled Reactions Between Pairs of Amino Acids induced by Cl. Sporogenes. Biochem. J., 30, 193k (1936). ~~ autobiography

I, Burk Allyn Dehority, was born on September 3,

1930* in Peoria, Illinois. 1 attended Loucks Grade School,

Columbia Jr. high School, and Woodruff high School all of

that city. in September of 191)8 i enrolled at Blackburn

College in Carlinvillc, Illinois, and upon completion of

my course work received an A.B. in chemistry in 1952.

Prior to graduation I had accepted a graduate assistant-

3hip in biochemistry at the University of Maine, and 1

received an M.S. degree from this institution in 1951).. l entered graduate school at Ohio State University in the

Department of Agricultural Biochemistry in October of 19 While completing the requirements for the degree of Doctor of Philosophy, 1 have held appointments as a Research

Assistant, a Research Fellow and an Assistant Instructor in the Department of Animal Science at the Ohio Agricultural Experiment Station.

1 0 2