S. Ratner

ENZYMES OF ARGININE METABOLISM IN CHICKS.

Abstract Ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate cleavage enzyme, and arginase, but not carbamyl phosphate synthetase, have been found in the chick kidney in moderate amounts. None of these, except traces of arginase, have been found in chick liver. Relatively small amounts of the cleavage enzyme have also been found in pancreas, spleen, and intestinal tract. The distribution of these enzymes is consistent with the ability of the chick to utilize citrulline in the diet in place of arginine and the inability to utilize ornithine in place of arginine. The significance of the findings is discussed in relation to arginine and nitrogen metabolism in the growing chick and in the developing embryo.

Enzymes of arginine metabolism in brain.

Abstract Argininosuccinate synthetase, argininosuccinate-splitting enzyme, and arginase have been found in the brains of all species investigated. The ability of this organ to synthesize arginine from citrulline is thus seen to take the same adenosine triphosphate (ATP)-dependent, two-step pathway as liver and kidney. The levels of activity found for a number of mammalian species, including man, were comparable to other enzymes of amino acid metabolism in brain. The implications of these findings are discussed in relation to the inborn error of metabolism associated with the disease known as argininosuccinic aciduria discovered by Allan et al. (1). A sensitive colorimetric method is described for the estimation of argininosucinate and is applied to the estimation of synthetase activity.

A study of ornithine, citrulline and arginine synthesis in growing chicks☆

Abstract Citrulline-C 14 , labeled in the ureido carbon, was fed to chicks maintained on a low arginine diet. About one-third of the isotope given was found in the amidine carbon of arginine isolated from tissue proteins. The results provide direct evidence that the chick does convert citrulline to arginine. Taken in conjunction with previous enzymatic findings, it becomes evident that the kidney and other extrahepatic tissues carry out the conversion and that the pathway followed is through argininosuccinate formation as with mammalian species. Further data indicate that the chick cannot convert ornithine to citrulline. This was shown by the absence of C 14 in tissue arginine after the administration of Na 2 C 14 O 3 together with unlabeled citrulline and a low arginine diet. Thus the indispensability of arginine in fowl nutrition and the absence of an ornithine cycle are both manifestations of the absence of the four enzymes of arginine synthesis from the liver. A new method for the isolation of ornithuric acid was applied to the investigation of ornithine synthesis. Evidence that the chick does not synthesize ornithine was shown by the absence of C 14 in the excreted ornithuric acid following the administration of sodium benzoate and glutamate-C 14 or Na 2 C 14 O 3 on a normal diet, or Na 2 C 14 O 3 on a low arginine diet.

Biosynthesis of guanidinoacetic acid. I. Purification and properties of transamidinase.

Abstract A transamidinase which catalyzes the reversible reaction, l -arginine + glycine guanidinoacetic acid + l -ornithine, has been obtained from hog kidney and purified about 80-fold. Ornithine exerts a strong product inhibition which influences the reaction rates; conditions are given for obtaining initial rates. The velocity of the forward reaction is six times faster than the reverse reaction, and the equilibrium constant has been found to have a value of 1.1. The position of equilibrium indicates that the “energy level” of the CN bonds in arginine are the same for all other guanidino amino acids. A weak hydrolytic activity toward arginine appears to be associated with the enzyme, as well as the ability to utilize l -homoarginine as amidine donor at a relatively low rate. A number of other properties of the enzyme are described.

Biosynthesis of guanidinoacetic acid. II. Mechanism of amidine group transfer.

Abstract Purified transamidinase has been shown to catalyze the transfer of the amidine group from the donors arginine, guanidinoacetic acid, and canavanine to the acceptors glycine, ornithine, and canaline. All possible donor-acceptor pair combinations can interact at appreciable rates. The results of isotope incorporation studies and of kinetic studies of product inhibition are consistent with a mechanism of amidine group transfer by direct interaction of donor and acceptor on the enzyme surface without the intermediate formation of an enzyme-amidine compound. The hypothesis proposed requires that the substrates be attached to the enzyme at adjoining sites in the catalytic area, that an acceptor molecule be capable of occupying either an acceptor site or the site occupied by the acceptor moiety of the donor molecule, and, conversely, that a donor molecule be capable of occupying both a donor and acceptor site. Competitions for the enzyme have been demonstrated between two acceptors, between an acceptor and a donor, and between two donors. It is suggested that where direct proof for the existence of an enzyme-compound or complex has not or cannot be obtained, inhibition analysis may serve to distinguish between a direct interaction and a successive-step mechanism.

Behavior of purified arginine desiminase from S. faecalis

Abstract Arginine desiminase from S. faecalis has been purified to the extent that 1 mg. catalyzes the hydrolytic cleavage of arginine to citrulline and NH 3 at the rate of 2500 μmoles/hr. (4200 moles/min./100,000 g. at 38 °), proceeding readily to virtual completion. A number of properties of the purified enzyme have been described. Direct evidence of phosphate involvement or of trace-metal ion or pyridoxal phosphate participation in the reaction mechanism has thus far not been obtained.

Argininosuccinase from bovine kidney: Comparison of catalytic, physical, and chemical properties with the enzyme from bovine liver

Abstract Argininosuccinase from bovine kidney has been prepared in crystalline form and compared to bovine liver argininosuccinase. The catalytic behavior, rate of inactivation (dissociation into subunits) at low temperature, and the sedimentation coefficient are the same. The antigenic properties are identical as examined by double-diffusion techniques. Antibodies to either one of the enzyme preparations are equally effective as inhibitors of the enzymatic activity. Comparison of the amino acid composition shows close agreement for most of the amino acids as well as a few minor differences. Considering the similarities in primary structure and in physical, catalytic, and antigenic properties, the argininosuccinase proteins from the two sources appear to be identical.

A new radiochemical assay for argininosuccinase with purified [14C]argininosuccinate☆

Abstract A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with 14C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-14C]arginine formed is measured as the sum of radioactivity in [U-14C]ornithine and [14C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 m m acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [14C]Argininosuccinate was synthesized enzymatically from l -[U-14C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions.

The dynamic state of body proteins.

MY TITLE “The Dynamic State of Body Proteins” would have been chosen by Rudolph Schoenheimer. It is closely linked to his famous book T h e Dynamic State of Body Constituents1 where he presented, with remarkable clarity, his new concepts on the dynamic state of the body fats, sterok, and proteins. This volume comprised the three Dunham Lectures given at Harvard in 1941. Up to that time the components of body tissues were regarded as being structural in function and metabolically inert. Schoenheimer applied the term “dynamic state” to some of the most complex constituents of the cell about which very little was known. The new findings and novel concepts described in those lectures were the results of experiments conducted over six or seven years in which the stable isotopes deuterium and 15N were applied to the study of intermediary metabolism by ingenious labeling of fatty acids, cholesterol, or amino acids. By chemical means the isotope was introduced into selected, chemically stable, locations of the molecule. This made it possible to follow the fate of a suspected metabolite in the living animal and to detect its participation in a multiplicity of hitherto unrecognized reactions. I was associated mainly with the studies on amino acid and protein metabolism. The material I plan to present might equally well bear the title “The Discovery of Protein Turnover” or in current parlance “The Molecular Basis for Protein Turnover.” The application of isotopes to the study of intermediary metabolism was an entirely novel undertaking and required a new methodology. It is appropriate to acquaint you with the circumstances under which the first tracer experiments of this kind began. Following on Harold Urey’s discovery in 1932 of the stable isotope deuterium, the Rockefeller Foundation, through Urey’s enthusiasm, undertook to support the exploitation of deuterium for biological studies. For this purpose, Rittenberg, who had been Urey’s student at Columbia and was trained by him in isotope techniques, came to the Department of Biochemistry at the Columbia College of Physicians and Surgeons (P & S) in 1934 to see what interest he could find. Hans T. Clarke, a highly gifted organic chemist and an influential proponent of the ap-

Argininosuccinase from bovine brain: Isolation and comparison of catalytic, physical, and chemical properties with the enzymes from liver and kidney

Homogeneous argininosuccinase has been isolated from bovine brain: compared to liver and kidney argininosuccinases from the same species, the catalytic activity (1400 U/mg). molecular weight of the fully active form (202,000 by gel filtration), and the minimum molecular weight (50, 000 in sodium dodecyl sulfate and mercaptoethanol) were in agreement with published liver and kidney enzyme values from this laboratory. That the brain enzyme is composed of four identical, or closely similar, polypeptide chains is supported by peptide maps analyzed after tryptic or cyanogen bromide cleavage. One-fourth the number of peptide fragments were produced as compared to the total number of susceptible residues per mole. The number of peptides containing other specific residues, or methionyl residues, were consistently one-fourth of the total considered. As maps of peptide fragments prepared from the brain enzyme were also superimposable, or nearly so, on liver enzyme maps, the four polypeptide chains from both sources were closely similar to each other in amino acid sequence. Distribution of the 16 sulfhydryl groups, as based on titration with Ellmans reagent, was in accord with the liver enzyme: Four sulfhydryl groups reacted without affecting catalytic activity, a second group of 4 became accessible on cold dissociation of the tetramers to catalytically inactive dimers, and the final 8 became accessible in strong dissociating agents. On analysis, Km values and negative homotropic interactions with substrate were in accord with liver enzyme kinetics. Immunological studies indicated a ciose resemblance in antigenic properties. The brain enzyme, as antigen, was fully crossreactive in the formation of precipitin bands with rabbit antibody to either liver or kidney enzymes already known to be mutually cross-reactive. The antibody to the liver enzyme was an effective inhibitor of brain enzyme activity comparable to inhibition of the homologous liver and kidney antigen.