Transamination in Tumors, Fetal Tissues, and Regenerating Liver* Philip P

Home , Oxaloacetic acid, Pyruvic acid, Transamination

Transamination in Tumors, Fetal Tissues, and Regenerating Liver* Philip P. Cohen, M.D., and G. Leverne Hekhuis

(From the Laboratory o~ Physiological Chemistry, Yale University School of Medicine, New Haven, Connecticut) (Received for publication June 9, I94I)

von Euler and co-workers (22), on the basis of TISSUE SOURCES results of essentially qualitative experiments, first re- Tumors.--The mouse tumors employed in this in- ported that the transaminase activity ot~ tumors was vestigation were the following: low. These investigators, using Jensen sarcoma and normal muscle, measured the rate of disappearance of I. United States Public Health Service No. ~7-- oxaloacetic acid in the following reaction: originally described as a neuroepithelioma (20). 1 2. Sarcoma 37 .1 3. Yale No. xuan estrogen-induced ,) 1( + )-glutamic acid + oxaloacetic acid a mammary adenocarcinoma (5). 1 4. No. x5o9I-A - ~'b a-ketoglutaric acid + aspartic acid spontaneous mammary medullary adenocarcinoma In addition to studies of reaction I in tumors, Braun- (6) "1 5- No. 42--glioblastoma multiforme. 2 6. No. stein and Azarkh (2) studied the reaction: i o8--rhabdomyosarcoma. 2 The tumors were transplanted at regular intervals 2) glutamic acid+a-keto acid to insure a uniform supply. a-ketoglutaric acid + amino acid "-b- Fetal, kitten, and adult cat tissues.--Two pregnant cats provided the fetal tissue. The length of the preg- The amino acids investigated in the case of reaction nancy was uncertain, but was estimated to be in the 2b were the d- and l-forms of alanine, valine, leucine, last trimester in both instances. Hemihysterectomies and isoleucine. The rate of reaction za in which both were performed under nembutal anesthesia and 2 d(--)- and l(+)-glutamic acid were used, plus fetuses removed from each animal. The mothers were pyruvic acid, was also studied. Braunstein and allowed to recover. One animal aborted the following Azarkh (2) reported very low rates of transamination day. The other animal delivered what appeared to be in a series of tumors, in some instances finding no full term kittens 8 days later. These were allowed to evidence of transamination, and in the others, usually nurse for 6 days. less than *5 per cent in 2 hours. These findings were Regenerating rat liver.uSeven albino rats, ,5 o to interesting since Braunstein and Azarkh reported rela- 18o gm. in weight were partially hepatectomized under tively fast rates for the same reactions in normal tissues. ether anesthesia (, i). In all instances, 6o to 7 o per cent However, it has been recently shown (8) that the rates of the liver was removed. The excised tissue was used of reactions 2a and 2b are very slow in normal rat for control determinations of transaminase activity. tissues, and that quantitatively the chief transamination The animals were then allowed to survive for varying substrates are those shown in reaction ,a. lengths of time, and the transaminase activity measured Since some metabolic characteristics of tumors are in the regenerating liver. also shared by embryonic tissue, it appeared of interest Transaminase activity in different species.~The to study transamination in such tissues. In addition, transaminase activity of liver, kidney, and skeletal regenerating liver was studied, since this represents a muscle of mouse, rat, and cat are of the same order rapidly growing tissue and thus might be expected to of magnitude; the per cent transamination in liver of show some metabolic similarity to tumor and embryonic tissue. x Transplants of tumors No. 17, sarcoma 37, Yale No. I and In this paper experiments are reported in which the i5ogi-A were obtained from Dr. M. Belkin, Department of Pa- rates of reactions , and 2 were studied quantitatively thology, Yale University School of Medicine. The authors are in 6 different mouse tumors. The rate of transamina- indebted to Dr. Belkin for his assistance in providing these transplants and suitable strains of mice for carrying all these tion with reaction ,a was also investigated with fetal, tumors. kitten, and adult cat tissues, in addition to regenerating 2 Transplants of tumors No. 42 and No. xo8 were obtained rat liver. through the kindness of Drs. H. M. Zimmerman and H. Arnold, of the Department of Pathology, Yale University School of * This investigation was aided by a grant from The Jane Medicine. These tumors were originally produced by intra- Coffin Childs Memorial Fund for Medical Research. cranial implantation of pellets of methylcholanthrene. 620

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1941 American Association for Cancer Research. Cohen et al.--Transamination in Tumors 62I these 3 species under the same experimental conditions a measurable transaminase activity when glutamic acid was found to be rat, 5o; mouse, 46; and cat, 47. plus oxaloacetic acid serve as substrates. Previous studies with purified transaminase (7) METHODS AND PROCEDURES showed that the per cent transamination varied with the square root of the enzyme concentration. On this Experimental procedure.--The tissues were removed basis it would be expected that if a large amount of from the animals after exsanguination and kept in ice- transaminase were present in a tissue, serial dilution cold saline. The tumors were dissected free of necrotic would not show this relationship until a concentration tissue. It was usually necessary to pool several tumors of transaminase was reached which was just sufficient for a given experiment. Suitable samples were then to catalyze reaction Ia at optimum speed. Below that weighed, after drying on filter paper, and homogenized concentration the per cent transamination would be (18) with M/Io phosphate buffer, at pH 7.4- This procedure was carried out at ice bath temperature. expected to fall according to the square root relation- Aliquots of the homogenized preparations were then ship. However, if the transaminase content of the pipetted into the reaction flasks. All experiments with tumors was low to begin with, it would be expected tumor tissue were carried out under anaerobic condi- that in addition to an initial low rate, the per cent tions, maintained by means of nitrogen gas and yellow transamination ,would decrease with dilution by an phosphorus, in order to rule out aerobic reactions. In the case of the other tissues, anaerobic conditions were TABLE I: ~-KETOGLUTARIC ACID FORMATION FROM not necessary since tissue dilutions of ::8o were used. L('~-)-GLUTAMIC ACID AND OXALOACETIC ACID All incubations were carried out at 380 C. with Microliters a-ketoglutaric acid found shaking. A Analytical methods.~In the experiments with the o o tumors, a-ketoglutaric acid was determined by the method of Krebs (i5) , pyruvic acid according to : + "~ ~+~ e, r ~.~ Westerkamp (23), and glutamic acid by a procedure Tumor --~ O _ previously described (4). The successful application No. 42 ...... xo5 54 670 471 35 of these analytical procedures to the measurement of No. I5O9I-A ...... 72 41 478 365 :'7 transamination has been previously reported (5-7). In No. :7 ...... 50 22 443 371 28 the case of the other tissues, reaction la was followed Yale No. I ...... 77 39 576 460 34 by measuring aspartic acid formation (6). S-37 ...... 98 41 624 485 36 Substrates.~The substrates were all brought to pH No. 1o8 ...... 65 52 514 397 3 ~ 7.4 before addition to the homogenized tissue, with the Each flask contained 3 ml. of homogenized tumor, dilution i:io, o.3 ml. of o.2 M l(+)-glutamic acid, and 0.3 ml. of 0.2 M oxalo- exception of the amino acids used in the experiments acetic acid added as indicated. Incubation time, I5 minutes; N2; shown in Table IV. In these experiments the amino yellow P; 38 ~ C. acids were weighed directly into the cups, the necessary amount of alkali, or acid, was added to neutralize, and amount determined by the square root of the dilution then M/3 phosphate buffer, pH 7.4, added to make factor. In Fig. : dilution curves are shown for the up a volume of o. 3 ml. The final concentration of the different tumors. The low initial rate and the rate of substrates added was o.o:6 M for each, except in the decrease of per cent transamination with dilution is case of dl-methionine, which was o.o32 M. In the seen to follow the square root relationship, indicating remaining experiments the substrate concentration was an initially low transaminase content. In contrast to O.Ol 4 M. this, most normal tissues show a much higher trans- The source and preparation of the different sub- aminase content (8). strates has been previously reported (6). Transamination with reaction 2a.--The per cent transamination with reaction 2a, in which the a-keto- acid was pyruvic acid, is shown for the different tumors RESULTS WITH TUMORS in Tables II and III. The data shown in Table II are Transamination with reaction xa.~In Table I typi- from experiments in which the disappearance of cal results are presented for a-ketoglutaric acid forma- pyruvic acid was measured. As can be seen, there is tion by tumors according to reaction ,a. The per cent no appreciable transamination with this system even transamination values for the 6 tumors are consid- after 60 minutes incubation and with a tissue concen- erably below those found for normal tissues, with the tration of i:io. Some of these experiments were re- exception of testis, lung, and spleen (8), but indicate peated and a-ketoglutaric acid formation was mea- that at high tissue concentrations (::1o) tumors show sured. As seen from Table III, in confirmation of the

1:7. o 40 o S-37 0 IZ v #42 E x Yale*l o 30 t a #1 08 o I.. 9 *'17 t-.- 9 *15091-A 20

0 I:10 1:40 1"80 DIlu'l',on FIG. i.--Transaminase activity of tumors at different dilutions. Substrates, l(+)-glutamic acid plus oxaloacetic acid o.o14 M. Incubation time, 15 minutes; N,.,; yellow P; 38.~ C. data in Table II, there is no significant formation of Transamination with reaction 2b.--The formation a-ketoglutaric acid. of glutamic acid from a-ketoglutaric acid and different amino acids was determined in 3 mouse tumors. As 'YABLE lI: TRANSA1VIINATIONWITH L(-ql-)-GLUTAMIC ACID PLUS can be seen from Table IV, the glutamic acid forma- PYRUVIC ACID (PYRuVIC ACID DISAPPEARANCE)

Pyruvic acid found after TABLE Ill: TRANSAMINATION WITH L(-~-)-GLUTAMIC ACID PLUS incubation PYRUVIC ACID (o.-KETOGLUTARIC ACID FORMATION) f A Pyruvic acid Microliters a-ketoglutarie Pyruvie plus acid found acid, 1 (+)-glutamic A Per cent micro- acid, micro- transami- r 1(+)- ' Tumor liters microliters liters nation Glntamic No. 42 ...... 1,285 1,24o 45 3 l(+)- acid plus Per cent Glutamie pyruvic transami- No. I5o9I-A .... 1,250 1,235 15 I Tumor acid acid A nation No. 17 ...... 1,33 ~ 1,3oo 30 2 No. 17 ...... 66 97 31 2 Yale No. 1 ...... 1,27o 1,225 45 3 No. 42 ...... 74 90 16 I S-37 ...... 1,26o 1,215 45 3 No. lO8 ...... 44 77 33 2 No. lO8 ...... 1,3oo 1,275 25 2 Each flask contained 3 ml. of homogenized tumor, dilution I:~ 9, o.3 ml. of o.2 M l(+)-glutamie acid and o.3 ml. of o.2 M pyruvtc Each flask contained 3 ml. of homogenized tumor, dilution i:io, acid added as indicated. N,~; yellow P; 38 ~ C.; incubation time plus ~ ml. of o.o6 M pyruvie acid, o.3 ml. of o.2 M l(+)-glutamic 6o minutes. acid added as indicated. N2; yellow 1:'; 38 ~ C.; incubation time, 6o minutes. tion is considerable only in the case of aspartic acid. In normal tissues, reaction 2a proceeds at a slow but When compared with most normal tissues (8), the measurable rate (8) when compared to reaction ia. In activity of reaction Ib in these tumors is also low. the case of these tumors it is apparent that reaction 2a Since reactions 2a and 2b have been shown to take does not take place at all, while reaction ~a proceeds at place at very slow rates in normal tissues (8), it is not a much slower rate than in normal tissue. surprising to find that these reactions do not proceed

to any appreciable extent in tumor tissue, inasmuch RESULTS WITH CAT TISSUES as the fastest reaction in normal tissues, reaction Ia, has been shown to take place at a slow rate in tumor Transamination in letal, kitten, and adult cat tis- tissues. sues.--In Fig. u data on transaminase activity, as Transamination with nonnatural amino acids.-- measured by reaction za, are presented for fetal, kitten, Braunstein and Azarkh (2) reported that d-amino and adult cat tissues. The fetuses were removed by acids show some activity with tumor tissue in the hemihysterectomy 8 days prepartum, as determined presence of a-ketoglutaric acid. However, the analyti- by the date of delivery of the remaining fetuses. The cal method employed by these workers was not satis- newborn kittens were allowed to nurse for 6 days factory for measuring small amounts of glutamic acid before they were killed. The mother cats served as as pointed out by Zorn (24). Inasmuch as reactions the sources of adult cat tissues. Transaminase activity 2a and 2b have been shown in this study not to proceed was measured in kidney, liver, and brain. at a significant rate with the l-amino acids it would As can be seen from Fig. 2, the per cent transamina- hardly be expected that the d-forms would be active. tion rapidly rises from fetal to adult tissues. Liver However, since reaction ia has been shown to proceed shows the greatest increase. The adult cat values for at a measurable rate in tumors, it seemed that d(-)- liver are of the same order as those for rat, while those glutamic acid would be more suitable for studying for kidney and brain are somewhat lower (8). The

TABLE IV: GLUTAMIC ACID FORMATION FROM o,-KETOGLUTARIC ACID AND DIFFERENT AMINO ACIDS

Glutamic acid found, Increas_e due to added amino Per cent microliters acid, microliters transamination r Jr_ sv ~. Yale No. Yale No. Yale No. Amino acid No. lO8 No. T I5O9I-A No. IO8 No. I I5O9I-A No. lO8 No. I I5O9I-A None ...... I65 131 12I ...... l(--)-Aspartic acid ...... 357 273 270 192 142 149 14 II II l(+)-Alanine ...... 2o5 167 191 40 36 70 3 3 5 1 (--)-Phenylalanine ...... 2oi 13o 81 46 ...... 3 .... dl-Methionine ...... 14 ~ 124 145 ...... 24 .... 2 1 (+)-Argininc ...... 270 81 136 42 . 9. 15 3 . . I I (+) -Tryptophane ...... 147 94 I22 ...... I ......

Each flask contained 3 ml. of homogenized tumor, dilution i:io, plus 0.3 ml. of 0.2 M a-ketoglutaric acid. Amino acids weighed into flasks in amounts equivalent to 0.3 ml. of o.a M (0.4 M for dl-amino acids) solutions. Incubation time 60 minutes; N.,; yellow P; 38 ~ C.

transamination of d-amino acids, yon Euler and co- per cent transamination values for fetal cat tissues are workers (22) using qualitative tests previously reported of the same order of magnitude as those found for that d(-)-glutamic acid was transaminated more mouse tumors. slowly with oxaloacetic acid in ]ensen sarcoma than Transamination in regenerating liver.--Data for the in normal muscle. transaminase activity of regenerating liver are shown

TABLE V[: TRANSAMINATION IN REGENERATING RAT LIVER TABLE V: TRANSAMINATION OF D(--)-GLUTAMIC ACID Days after Mgm. dry Per cent Microliters a-ketoglutaric acid found partial weight of transamina- Qtransamina- hepatectomy liver tion tion d ( - ) -Glut amic plus o II. 5 52 243 Tumor d (-)-Glutamic Oxaloacetic oxaloacetic 2 IO.2 4 ~ 212 S-37 ...... 73 42 85 5 lO.O 39 21o No. 42 ...... 66 63 102 9 It.2 35 I68 No. I5O9I-A ...... 63 76- 96 12 11, 5 51 238 Each flask contained 3 ml. of homogenized tissue, dilution i:,o, 24 II.6 53 245 0.3 ml. of o.2 M d(-)-glutamie acid and 0.3 ml. of 0.2 M oxaloacetic acid added as indicated. Incubation time, 6o minutes; Substrates, l(+)-glutamic acid plus oxaloacetic acid (equimolar 38 ~ C.; N.,; yellow P. concentration, o.o14 M). Tissue dilution, 1:8o; incubation time, 15 minutes; air; 38 ~ C.

Quantitative measurements of this reaction are in Table VI. Transamination in these experiments shown in Table V. As will be noted, in none of the was measured with the substrates 1( + )-glutamic acid 3 tumors studied was there any evidence of transamina- plus oxaloacetic acid (reaction Ia). As can be seen tion with d(--)-glutamic acid. This finding is in from the data, there is a significant fall in per cent keeping with previous experiments with purified transamination between 5 and 9 days after partial transaminase preparations (6) and pigeon breast hepatectomy, after which the per cent transamination muscle (5). returns to a normal value.

The ~ttransamination values are also decreased sig- TABLE VII: QTRANSAMINATION nificantly, indicating that the fall is not due to an Tumors Fetal tissues (eat) Adult tissues (cat) increase in water content of the livers. These experi- S-37 ...... 57 Kidney .... 78 Kidney .... 220 ments, while not striking, fall in line with those for No. 42 .... 72 Brain ...... 77 Brain ...... 21o tumor and fetal cat tissues, and suggest that where Yale No. I.. 54 Liver ...... 64 Liver ...... 230 No. x5o9I-A. 54 Placenta . 9 9 94 growth is taking place rapidly, the transaminase No. Io8 ... 72 activity is decreased. No. I7 .... 53 6C

~4C 0 C E ~3C C O k.

x 2O

I0 -~K B L K B L K 0 Fetus Kitten Cat Fxc. 2.---Transaminase activity in fetal, kitten, and adult cat tissues. Substrates, l(+)-glutamic acid plus oxaloacetic acid, o.oI 4 M. Tissue concentration z :80; incubation time, I5 minutes, air; 38.~ C.

Qtransamination.--The rates of transamination in DISCUSSION the different tissues can best be compared in terms of Qtransamination. Tumor, fetal cat, and regenerating liver tissue have in common the property of rapid growth, or rapid Qtransamination = protein synthesis. These tissues, particularly the first microliters o[ substrate transaminated two, also show a low transaminase activity. This would mgrn. dry weight • hours suggest that an inverse relationship exists between In Table VII are listed Qtransamination values for protein synthesis and transaminase activity. The exact mouse tumors, fetal cat, and adult cat tissues. It is mechanism by which the transamination reaction could seen that the Qtransamination values for the fetal and influence protein synthesis is not clear. However, if tumor tissues are of the same order of magnitude. one considers the substrates involved in reaction I, However, the values for the adult tissues are several it is seen that we are dealing with substances which times greater than those for the corresponding fetal are known to play special roles in intermediary metab- tissues. The ~transamination value for regenerating olism. Thus, oxaloacetic acid and a-ketoglutaric acid rat liver 9 days after partial hepatectomy (Table VI) act as respiratory mediators in the Szent-Gyorgyi- is of the same order of magnitude as that of kitten Krebs cycle (i6). On the other hand, glutamic acid liver, but apparently never reaches the low value of and aspartic acid play important roles in protein fetal cat liver. metabolism. Schoenheimer and Rittenberg (i9) have

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1941 American Association for Cancer Research. Cohen et al.--Transamination in Tumors 625 stated, "The results obtained with N ~ strongly support The recent finding that tumors and embryonic tissue the theory that the dicarboxylic acids play a central are low in arginase (IO) may represent another mecha- role in protein metabolism." These workers observed nism by which an amino acid is "preserved," as it were, that whenever isotopic amino acids, or ammonia, were for the purpose of protein synthesis in rapidly growing fed, the dicarboxylic acids showed a much higher tissues. isotope concentration than the other amino acids, with SUMMARY the exception of the amino acid fed. Glutamic acid always showed a higher concentration of isotope than 1. The transaminase activity of a series of 6 mouse aspartic acid. tumors, regenerating rat liver, and fetal kitten and While the facts are too few to permit a satisfactory adult cat tissues was determined with the reaction: forn-mlation of the mechanism by which transamina- I) l(+)-glutamic acid+oxaloacetic acid tion influences protein synthesis, for purposes of a a-ketoglutaric acid + aspartic acid working hypothesis the interrelationship might be "-b- pictured as follows. Glutamic acid plays a central role The tumors and fetal tissues showed low activities in protein synthesis. From the work of Schoenheimer and the regenerating liver a somewhat lowered activity and Rittenberg (i9) it is clear that the protein mole- when compared to normal adult tissues. cule cannot be considered as a stable substance, but 2. No transaminase activity was observed with three rather as a highly reactive compound which con- different tumors when d( -- )-glutamic acid was used tinuously participates in exchange reactions with free in reaction ia. amino acids. Thus the protein molecule can be pic- 3. The reaction: tured as existing in a dynamic equilibrium with free 2) l(+)-glutamic acid+a-keto acid amino acids and other metabolites. In the presence of "T-~ a-ketoglutaric acid + amino acid high concentrations of transaminase, the amount and rate of protein synthesis would be determined by the was also studied with different tumors. In the case rate at which glutamic acid is "pulled out" of the of reaction 2a, with pyruvic acid as the a-keto acid, protein-amino acid equilibrium system. Since reaction no measurable transamination occurred. In the case ia proceeds at a rate 2 to 3 times as fast as reaction Ib of reaction 2b, the amino acids l(-)-aspartic acid, (7, 8), aspartic acid synthesis would take place at the l(+)-alanine, l( -- )-phenylalanine, dl-methionine, expense of glutamic acid, with the result that protein 1( + )-arginine and i( + )-tryptophane were used. The synthesis would be limited. The high transaminase transaminase activity was appreciable only in the case activity of adult tissues with a slow rate of protein of l( -- )-aspartic acid (reaction Ib). synthesis, and the low activity of tumor and embryonic 4. The suggestion from these experiments that an tissues with a high rate of protein synthesis, fit into inverse relationship exists between transaminase ac- such a hypothesis. tivity and protein synthesis is discussed. In addition to low transaminase activity, low values REFERENCES for cytochrome C (12, 17, 2I), coenzymes I and II I. BERNHEIM, F., and FELSOVANYI,A. V. 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Philip P. Cohen and G. Leverne Hekhuis

Cancer Res 1941;1:620-626.

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