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d -(−)-Arginine was isolated by fractionally recrystallizing dl -arginine- l s -(−)-malate to obtain the less soluble d -(−)-arginine- l s -(−)-malate, then passing aqueous solutions of the latter through ionic resin columns to separate the d -arginine from the malic acid. The d -arginine was converted to the monohydrochloride. The fractional crystallization readily yielded 51–73% of the d -arginine- l -malate. d -Arginine monohydrochloride was obtained from the latter in yields of 74–83% of the theoretical. Impure l -arginine was separated from the more soluble arginine malate residues. Two methods of racemizing it for recycling were tested. The separated malic acid was evaporated to dryness in vacuo for reuse, or precipitated as the calcium salt.

Despite the markedly increased excretion of urocanic acid induced in young male rats upon supplementing their 12% casein diets with 0.05% of dl -ethionine, their rate of growth was not appreciably retarded after the first 4 days. The ethionine markedly suppressed the activity of the liver urocanase, but had little effect upon the activity of hepatic histidine deaminase. The possibility that feeding extra l -histidine might increase the activities of histidine deaminase and urocanase was not substantiated. Similar increases occurred in normally fed controls. The levels of liver glycogen in control animals previously fed differing, but normal basal diets, varied with the diet, but the extra glycogen deposition produced by the administration of l -histidine was nearly the same in all. The net deposition after the administration of urocanic acid was much smaller and of questionable significance. Slow absorption of urocanic acid from the alimentary tract was at least partially responsible. Ethionine-fed rats produced little or no liver glycogen, whether given histidine, urocanic acid, or glutamic acid. Such behavior is consistent with the suggestion that ethionine may trap sources of high energy, some of which are known to be required for glycogen synthesis.

Even when fed in a diet which contains all of its other amino acids in the l form, d -valine promotes growth so poor as to indicate extremely inefficient inversion. Under similar circumstances, d -leucine undergoes inversion readily enough to produce moderate, though definitely subnormal growth. Replacement of the l -leucine in the d -valine diet by twice as much dl -leucine impaired the inversion of d -valine enough to prevent growth, and the further addition of an equal amount of d -leucine depressed the growth still more. No inhibition of the inversion of suboptimal levels of d -methionine and d -phenylalanine could be detected in analogous tests. At a 2% dietary level, dl -norleucine prevented growth on either d -valine or d -leucine and retarded growth on complete l -amino acid diets. At a 1% level, neither dl -norleucine nor dl -norvaline affected the response to the l -amino acid diet. Each re tarded, but did not prevent the growth on d -valine or d -leucine. Implications of these observations are discussed.

Both in vivo and in vitro tests with radioactively labeled histidine and urocanic acid showed that rat liver tissue is more readily permeable to histidine than to urocanic acid. Total urinary output of urocanic acid and its radioactivity were measured after the administration of l -histidine by stomach tube and of urocanic acid by subcutaneous injection (one labeled; the other unlabeled) in rats which had previously been fed normal diets or diets supplemented with 0.05% of dl -ethionine. Labeling only the histidine or only the urocanic acid made it possible to determine the contribution of each to the urocanic acid output. In the normal animal both preformed urocanic acid which enters the cell and urocanic acid produced in it can be further metabolized. In the ethionine-fed animal further metabolism is limited by the inhibition of urocanase, and urocanic acid accumulates within the cell. The accumulation resists the entrance of extracellular urocanic acid, which in turn checks the excretion of cellular urocanic acid and its further production from histidine. The data provide a rational basis for reconciling the limited glycogen production induced by urocanic acid with the concept that urocanic acid is nevertheless a significant intermediate in the histidine-glycogen pathway.

In rats fed d -valine diets adequate in l -leucine, d -leucine supplements (i.e., twice as much dl -leucine) decreased the rate of growth, apparently by competing with d -valine for inversion. Doubling the l -leucine instead produced less retardation. α-Ketoisovaleric acid promoted as rapid growth as l -valine. α-Ketoisocaproic acid did not retard the response to d -valine and the addition of isovaleric acid did so only slightly. When the slow rate of growth induced by the d -valine was reproduced by substituting just enough l -valine, dl -leucine at twice the normal l -leucine level promoted less retardation than it did in the diets which contained the d -valine. Definite antagonism in response toward 1.4% of dietary d -valine was induced by 1.4% of d -norvaline, less by l -norvaline; 0.7% of d -norvaline was not noticeably antagonistic. Antagonism toward l -valine was questionable at both levels. In a d -leucine diet adequate in l -valine, d -valine retarded growth only moderately. Addition of l - or d -norleucine definitely inhibited the response to d -leucine, but only moderately the response to l -leucine. dl -Norleucine retarded the response to d -valine, α-ketocaproic acid was less inhibitory, and valeric acid produced no retardation. In vitro tests with purified d -amino acid oxidase supported surprisingly well the assumption that competitive inhibition of deamination is probably primarily responsible for the in vivo antagonisms noted.

Antagonism at the oxidative deamination stage may well be one of the factors responsible for the poorer growth observed when a d -amino acid requiring inversion is fed in a diet which contains a large proportion of other d -amino acids. The rate of oxygen uptake when two d -amino acids were incubated together with crude and purified extracts of hog kidney and with slices of rat kidney was less than the sum of the rates when each was oxidized separately. Data based upon the Chromatographic separation and estimation of the α-keto acids produced, or upon the spectrophotometric measurement of one keto acid, with calculation of the second from extra oxygen uptake, showed that the ratios of their degrees of involvement when they were oxidized simultaneously were not the same as their ratios when they were oxidized separately. Lineweaver-Burk treatment of data obtained with purified d -amino acid oxidase indicated that the inhibition was competitive. The maximum velocity of the oxidation of two amino acids jointly approximated that of the amino acid with the lower Michaelis constant.

It has long been known that Oidium lactis is able to utilize the nitrogen of several natural amino acids for growth when such acids serve as the sole source of nitrogen. Under such circumstances, l-tryptophane is converted into l-indolelactic acid.1 In conjunction. with a series of studies on tryptophane metabolism recorded elsewhere, in which Oidium lactis was used to synthesize indolelactic acid,2 it seemed of interest to compare the availability of l- and dl-tryptophane and acetyl-l- and acetyl-dl-tryptophane as sources of nitrogen for the growth of this mold.Accordingly, 8 liters of a synthetic medium containing 80.0 gm. invert sugar, 8.00 gm. K2HPO4, 0.800 gm. MgSO4, and traces of NaCl and FeCl3 were prepared and divided equally among eight 2800 cc. culture flasks. To each of one pair of the flasks were added 2.000 gm. of l-tryptophane; to each of a second pair, 2.000 gm. of dl-tryptophane; to each of a third pair, 2.412 gm. of acetyl-l-tryptophane; and to each of the fourth pair, 2.412 gm. of acetyl...

The d -amino acid oxidase in sonicates of Trigonopsis variabilis was purified by precipitating it with acetone and with ammonlum sulfate, removing nucleo-proteins with protamine sulfate, adsorbing impurities with charcoal, and applying gel and ion-exchange chromatography. The final fraction had a specific activity over 250 times greater than that of the initial sonicate. At 38°C, toluic and benzoic acids did not inhibit the enzyme appreciably, but heating to 55°C for 5 min completely inactivated it. Inhibition by p-chloromercuribenzoate and its partial reversal by 2-mercaptoethanol indicated the probable presence of functional sulfhydryl groups. Addition of FAD did not markedly enhance the activity of the purified enzyme, presumably because the FAD originally present was too tightly bound to permit excessive loss in the purification steps. The apparent Michaelis constant of the purified enzyme for d -leucine approximated 2.8 × 10−4 m . In descending order, activities toward the several d -amino acids tested were: d -leucine > d -tryptophan > d -methionine > d -histidine > d -alanine. The purified enzyme did not attack d -threonine.

To determine the influence of stereoisomerism upon the course of histidine metabolism, metabolite excretion in 24 hr was assayed in rats previously fed normal and ethionine-supplemented diets for 12 days, then fasted for 12 hr before being given normal or radioactive d - or l -histidine, subcutaneously or by mouth. When histidine-α-14C was given, contributions to carbon dioxide excretion and protein deposition were also measured. In rats fed normal diets, the greater outputs of histidine, imidazolepyruvic, imidazoleacetic, and imidazolelactic acids induced by d -histidine support its inversion via imidazolepyruvic acid, with imidazoleacetic and imidazolelactic acid produced secondarily by oxidative decarboxylation and reduction. The smaller yields of urocanic, formiminoglutamic, and glutamic acids from d -histidine undoubtedly reflect their production only from the l -histidine formed by inversion. The conclusions drawn are supported by the quantitative differences observed with l -histidine, by differences induced in ethionine-fed animals, and by those found in the carbon dioxide and protein assays. Metabolite separation was effected by thin-layer chromatography. Assays were made spectrophotometrically or by scintillation counting, or both. Specific activities of the metabolites indicated marked dilution only in the glutamic acid. Contributions to the carbon dioxide excreted and to the proteins precipitated were estimated by counting.

The gastrointestinal absorption of the d and l isomers of several amino acids was studied in rats by the Cori technique. In one series of tests each isomer was fed separately, and in a second series a dl mixture was employed in which one optical component was labeled and the other was unlabeled. In the first series, l -methionine underwent absorption more slowly than d -methionine, and l -aspartic acid insignificantly more slowly than d -aspartic acid. In similar tests of the l and d forms of valine, histidine, phenylalanine, and tryptophan, the l isomer was the more rapidly absorbed. When equal quantities of the l and d forms of methionine, valine, histidine, phenylalanine, and tryptophan, respectively, were mixed and fed, each isomeric component was absorbed more slowly than it had been when it was fed alone, but in every instance the l component was more rapidly absorbed than the d . Joint feeding with the l isomer of methionine slowed the absorption of l -tryptophan, l -histidine, and an amino acid mixture more so than did joint feeding with the d isomer. Absorption of the amino acid mixture was retarded more strongly by methionine than by tryptophan.