AMINO-ACID SYNTHESIS IN THE ANIMAL ORGANISM.

AMINO-ACID SYNTHESIS IN THE ANIMAL ORGANISM. CAN NOR-LEUCINE REPLACE LYSINE FOR THE NUTRITIVE REQUIREMENTS OF THE WHITE RAT? BY HOWARD B. LEWIS AND LUCIE E. ROOT. (From the Laboratory of Physiological Chemistry of the University of Illinois, Urbana.) (Received for publication, June 15, 190.) Of the possibility of amino-acid synthesis in the normal mammalian organism, little is definitely known. Perfusion experiments with the surviving liver (1) have shown that cu-keto- and a-hydroxy-acids may be converted to the corresponding aminoacids, to the optical isomer that exists in the protein molecule. The formation of tyrosine, phenylalanine, alanine, leucine, and nor-leucine among others has t.hus been demonstrated. In the living organism, however, the synthesis of a-amino-acids by the reaction between ammonia and non-nitrogenous rests cannot be regarded as proved, despite the many experiments () which have been undertaken with this in view. Of amino-acids present in the protein molecule, only glycocoll is known to be synthesized, and even in the case of this amino-acid no proof exists as to whether it takes its origin from glycollic or glyoxylic acid and ammonia, a direct synthesis of the type discussed above, or whether it arises from cleavage of some other amino-acid of higher molecular weight (3). Tyrosine may possibly be derived from phenylalanine (4), and histidine from arginine (5), but neither of these reactions involves an amination of a non-nitrogenous carbon chain. The present study is concerned with the formation of lysine by amination of the E carbon of nor-leucine. It seemed possible that, if the or-amino group were already present in the molecule, further substitution of a second amino group might take place more readily. The possibility also suggested itself that, even if this formation of lysine from nor-leucine did not occur, nor-leucine might 79

Nor-Leucine still have a value in nutrition. According to the current idea, the E group of lysine does not function in peptide synthesis and is present in the protein molecule free and not combined in the peptide linkage. The organism might have the power to synthesize a new type of protein under stress by the substitution of norleucine for lysine, a protein which would contain little or no free amino nitrogen, since it is to the E group of lysine that this free amino nitrogen of the protein molecule is usually attributed. The validity of the present experimental work is dependent upon the fact that lysine in adequate amounts is essential for the normal growth of the young white rat. Osborne and Mendel (6) have shown that gliadin with its low content of lysine as the sole source of the protein of the diet does not permit normal growth unless supplemented by lysine, while with zein (7), which is deficient in both tryptophane and lysine, as the source of protein maintenance results from the addition of tryptophane, but no growth unless lysine is also added. Hart, Nelson, and Pitz (8) were unable to demonstrate a synthesis of lysine by the mammary gland of the white rat, to supply an adequate milk for the young. Buckner, Nollau, and Kastle (9), and Osborne and Mendel (10) have also demonstrated that chickens require a sufficient amount of lysine to make normal growth. Nor-leucine, the third of the isomeric cr-amino-caproic acids known to exist in the protein molecule, was discovered by Abderhalden and Weil (11) in 191 in the proteins of the central nervous system. However, it is in all probability also present in proteins derived from other sources, but since its solubilities and general properties so closely resemble those of the other isomeric cr-amino-caproic acids as to make its separation from them difficult, its occurrence has as yet been reported in the proteins of the nervous system only. Greenwald (1) has demonstrated that nor-leucine behaves normally in the animal body, yielding its nitrogen as urea after ingestion. He has also observed that nor-leucine is a glucose-former in the phlorhizinized dog, and that the amount of glucose formed from the d, 1, or dl forms does not vary appreciably.

H. B. Lewis and L. E. Root EXPERIMENTAL. Young white rats of 50 to 60 gm. weight for the most part ~were used as experimental animals. They were kept in individual cages which permitted accurate record of the food consumption. The usual precautions to insure cleanliness and prevent infection were taken. It rnay be noted that only one case of disease, a type of pulmonary infection, appeared in the colony. The food which was high in fat content was made into small balls weighing 5 to 10 gm. each which were kept in an ice chest until used. In this form the food was not easily scattered by the animal and there was little wasting. The casein, g&din, nor-leucine, and lysine were prepared in the laboratory. The lysine was obtained by hydrolysis of casein, while the nor-leucine was a synthetic product. The protein-free milk prepared by the method of Osborne and Mendel contained 0.59 per cent nitrogen. There has been considerable criticism of the use of this subst.ance in nutrition experiments from time to time, but in the present series the objections offered seemed to be invalid, since the diet as a whole remained constant throughout with variations from the addition of small quantities of the amino-acids only. If there were any appreciable amounts of essential amino-acids furnished by the protein-free milk, these were present in each of the periods, and their influence should have been uniform. The experimental dietaries were as follows: per cent per cent Casein or gliadin... 18.0 Amino-acid............. 0.5 Protein-free milk........ 8.0 Gliadin................ 17.5 Corn-starch... 4.0 Protein-free milk........ 8.0 Purified butter fat..., 18.0 Corn-starch..............0 Lard................... 1.0 Purified butter fat..... 18.0-100.0 Lard.................... 14.0 100.0 Rats were maintained on diets containing 18 per cent gliadin for periods of several weeks; nor-leucine replaced part of the gliadin usually in amounts equivalent to 0.5 per cent of the diet, the protein plus amino-acid still constituting 18 per cent of the total ration; finally in some experiments equivalent amounts of lysine

8 Nor-Leucine replaced the nor-leucine. The relative order of these periods was varied, but the general procedure remained the same. Controls were fed on casein rations to demonstrate that normal growth was possible on these diets provided the protein was adequate. In some of the experiments, 1.5 per cent of nor-leucine was fed inst,ead of 0.5 per cent. Lysine to the extent of 0.5 per cent will supplement a diet containing 18 per cent gliadin such as was used in these experiments (6) and allow normal growth. The nor-leucine, however, was racemic and as it was considered desirable to insure an adequate amount of the d form in the diet, in case the animal could not use both forms equally well, the higher percentages were added to the ration. In later experiments through the kindness of Dr. C. S. Marvel of this laboratory, we were supplied with small amounts of d-nor-leucine, which showed on analysis 8 per cent of the d form. This was added to the diet to the ext,ent of 0.658 per cent equivalent to 0.5 per cent, of the d form. No difference could be observed between this optically active form and the racemic compound. DISCUSSION. The results of the experiments are given in Table I and Charts 1 and. Rats fed on casein grew normally in all cases.l The curves for Rats 5, 8, and 17 show the type of growth obtained when gliadin was substituted for casein. Slight growth ensued, a result which was in agreement with the findings of Osborne and Mendel (6) previously discussed. Rat 3 was fed a diet in which 0.5 per cent dl-nor-leucine replaced an equivalent amount of gliadin. The curve of growth for this animal closely resembles that for the rats fed gliadin alone. Animals which received a higher percentage of the dl-nor-leucine (Rats 6, 8, 14, 17) did not make any better growth than those fed 0.5 per cent. The cessat,ion of growth in older animals, which had been gaining in weight steadily on casein diets, when gliadin plus nor-leucine was substituted as the source of protein may be observed in the curves for Rats 11 and 1. In order to prove Ohat this failure to increase in weight on gliadin and nor-leucine was not due to the 1 The growth curves of the casein-fed controls are not shown in the charts, but Table I includes a record of their weights and food consumption.

H. B. Lewis and L. E. Root 83 TABLE I. Summary of Weights and Food Intake of Experimental Animals. Clasein. Gliadin. I Diet. (a) Gliadin. (b) + 0.5 per cent (a) Casein. (b) Gliadin + 0.5 per cent (a) Casein. (b) Gliadin + 1.5 per cent (a) Gliadin. (b) + 1 per cent lysine. (c) Gliadin + 0.5 per cent (a) Gliadin. (b) + 1.5 per cent (c) Gliadin + 0.5 per cent d-nor-leucine. (d) Gliadin + 0.5 per cent lysine. (a) Gliadin. (b) + 1.5 per cent (c) Gliadin + 1 per cent lysine. (d) Gliadin + 0.5 per cent. g - EazJs 98 84 56 91 91 8 Fj. $ 3 67m. 97 63 6 100 68 101.r $ it 777%. 50 190 06 117 88 101.-.! 6-7m..5.7 144 16 0 00 -i,o ;;i % B!lm. 377 316 153 $56 151 15 $j r. au, Fii 5 - am. 17.8 0.5 31.9 3.7 4.3 0.0 91 101 13 30 496 31 4.0 4 70 150 80 96 43 7.0 91 4 98 70 4 35 4 84 4 4 8 56 4 36 150 149-1.: 610 70 130 60 75 130 140 10 558 49 58 10 71 58 113 113 115 76 77 77 88 88 91 91 10 63 6 6 74 74 13 13 16 A- - 54.3 1 11 3 19-1 11 50 43 173 184 349 171 08 133 30 48 05 31 44 30 48 40 5 36 33 31 31 43 38 35 8-0..0 1.7 3.5. 1.3 0.5 3.1 1.7 9.3-0.7 4.8 0.0 1.

84 Nor-Leucine TABLE I-conclu&?d. s Diet. 4 C? (a) Casein. (b) Gliadin + 0.5 per cent (c) Gliadin + 0.5 per cent lysine. (d) Gliadin + 1.5 per cent 14 0 (a) Gliadin + 1.5 per cent (b) Gliadin + 0.5 per cent lysine. 15 0 (a) Gliadin + 1.5 per cent (b) Gliadin + 0.5 per cent d-nor-leucine. 17 0 (a) Gliadin. (b) + 1.5 per cent 63 106 35 118 35 167 84 6 35 6 56 57 4 57 56 57 63 59 inability of the organism to resolve and metabolize the racemic amino-acid, Rats 8 and 15 were fed d-nor-leucine in place of the racemic form. The results differed in no way from those of the preceding experiments. As a final check on the experimental procedures, lysine replaced the nor-leucine of t:he diet of Rats 4,5,6, 8, and 14 (Chart ). In confirmation of the work of Osborne and Mendel, this amino-acid supplemented the diet in such a way as to permit normal growth. On the addition of lysine, there was a marked improvement in the general condition of the rats previously stunted by a gliadin or a gliadin plus nor-leucine diet, an improvement which was noticeable within the 1st week. The increased rate of growth is not to be attributed to an increased food consumption, since as shown in the table the food eaten per 100 gm. of body weight in the lysine period was practi- 811. 106 118 167 160 6 97 57 65 59 6.E! 8 Q?n. 47 1 49 97 00 35 00 8 3 on. 140 397 49 34 81 143 199 110 4 185 34 9 4 35 35 44 30 50 34 @m. 33.7 cally the same as in the preceding nor-leucine period. The gains per 100 gm. of food consumed on the lysine-gliadin diets were com- 3.0 15.5-3.0 00.0 4.4 00.0 7. 0.9 1.6

E-c-a? T 0p. - CHART 1. Showing the failure to grow normally when dl-nor-leucine supplements an 18 per cent gliadin diet. The curves for Rats 11 and 1 also show the prompt cessation of growth in older and larger rats when casein is replaced by gliadin and nor-leucine. In the case of Rat 15 no improvement, results from the ingestion of the d-nor-leucine in place of the dl form. I

86 Nor-Leucine CHART. Showing that the deficiency in a gliadin plus nor-leucme diet may be remedied by substitution of lysine (dotted line) for nor-leucine, and that cessation of growth ensues when nor-leucine replaces lysine. The curve for Rat 8 also demonstrates that d-nor-leucine is no more efficient as a supplement to gliadin than is the racemic compound. parable to those made on the casein diets, while with gliadin or gliadin plus nor-leucine the gains in proportion to the food consumed were almost negligible. SUMMARY. 1. Gliadin comprising 18 per cent of the diet has been shown to be inadequate for normal growth of the white rat, and to permit, of maintenance or slow growth only. Lysine in ammounts equiva-

H. B. Lewis and L. E. Root 87 lent to approximately 3 per cent of the protein of the above diet without further alteration renders the diet adequate for normal growth. These results are in agreement with those of Osborne and Mendel.. Neither dl- nor cl-nor-leucine is able to supply the deficiency of a gliadin diet as does lysine. When nor-leucine is replaced by lysine in the experimental dietaries, normal growth ensues. 3. No evidence has been obtained which would indicate that nor-leucine is a precursor of lysine or that the organism of the white rat can substitute nor-leucine for lysine in the synthesis of its body protein for growth. BIBLIOGRAPHY. 1. Embden, G., and Schmits, E., Biochem. Z., 1910, xxix, 43; 191, xxxviii, 393.. Grafe, E., and Schlapfer, V.,. physiol. Chem., 191, Ixxvii, 1, and later papers by Grafe and collaborators. Abderhalden, E.,. physiol. Chem., 191, Ixxviii, 1, and later papers. Pescheck; E., Biochem. Z., 191, XIV, 44. Underhill, F. P., and Goldschmidt, S., J. Biol. Chem., 1913, xv, 341. 3. Magnus-Levy, A., Biochem. Z., 1907, vi, 541. Epstein, A. A., and Bookman, S., J. Biol. Chem., 191-13, xiii, 117; 1914, xvii, 455. 4. Abderhalden, E.,. physiol. Chem., 1915, xcvi, 1. Totani, G., Biothem. J., 1916, x, 38. 5. Ackroyd, H., and Hopkins, F. G., Biochem. J., 1916, x, 551. Geiling, E. M. K., J. BioZ. Chem., 1917, xxxi, 173. 6. Osborne, T. B., and Mendel, L. B., J. BioZ. Chem., 1914, xvii, 35. 7. Osborne, T. B., and Mendel, L. B., J. BioZ. Chem., 1915, xx, 351. 8. Hart, E. B., Nelson, V. E., and Pits, W., J. BioZ. Chem., 1918, xxxvi, 91. 9. Buckner, G. D., Nollau, E. H., and Kastle, J. H., Am. J. Physiol., 1915-16, xxxix, 16. 10. Osborne, T. B., and Mendel, L. B., J. BioZ. Chem., 1916, xxvi, 93. 11. Abderhalden, E., and Weil, A.,. physiol. Chem., 191, lxxxi, 07; 1914, lxxxviii, 7. 1. Greenwald, I., J. BioZ. Chem., 1916, xxv, 81.

AMINO-ACID SYNTHESIS IN THE ANIMAL ORGANISM: CAN NOR-LEUCINE REPLACE LYSINE FOR THE NUTRITIVE REQUIREMENTS OF THE WHITE RAT? Howard B. Lewis and Lucie E. Root J. Biol. Chem. 190, 43:79-87. Access the most updated version of this article at http://www.jbc.org/content/43/1/79.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/43/1/79.citation.full.html #ref-list-1