Shigeo Takada

Qualitative and quantitative abnormalities of argininosuccinate synthetase in citrullinemia

Enzymological and immunochemical analyses of the liver were preformed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlonys double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The Km values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher Km values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.

Crystallization and some properties of argininosuccinate synthase from rat liver.

In the urea cycle argininosuccinate synthase activity is rate-limiting [ 1,2] and may be a regulatory enzyme. While the other urea cycle enzymes have been purified to homogeneity [3,4,5,6], this enzyme has been purified from steer liver and hog kidney to only 50% purity [7,8] . Recently Ratner described in Advances in Enzymology [l] briefly that they succeeded in the crystallization of the enzyme from steer liver. In the present paper, the isolation, the crystallization and some properties of argininosuccinate synthase from the rat liver are described. The enzyme consisted of four subunits with identical mol. wt of 48 000 * 1000. Its catalytic properties such as optimal pH and Km values were similar to those of the enzyme from steer liver and hog kidney [7,9].

Role of argininosuccinate synthetase in the regulation of urea synthesis in the rat and argininosuccinate synthetase-associated metabolic disorder in man

Abstract Regulation of urea synthesis and the role of ASS were studied in rats subjected to acute dietary transitions from high to low protein or vice versa and in rats injected with an ammonium salt. Significant increase/decrease in urea excretion and urea in the liver preceded increase/decrease of ASS during the dietary transitions. Shortly after the switch of diet from high to low protein, the ratios of the rate of urea synthesis from ammonium chloride to the activity of ASS in the perfused liver decreased to much lower values than those of rats fed on the low protein diet. In contrast, during the acute transition from low to high protein diet, the ratios increased above the values of rats fed on the high protein diet. The results indicated that regulatory factors other than the activity of ASS may influence urea synthesis. The concentrations of ornithine and acetylglutamate capable of stimulating urea synthesis markedly changed shortly after the dietary switch. The fact that fluctuations of ornithine and acetylglutamate concentrations were greater and occurred prior to the activity changes of ASS strongly suggests a possible regulatory function of these amino acids in urea synthesis. ASS may be rate-limiting only during the dietary transition from low to high protein. Intraperitoneal injection of an ammonium salt caused an increase in ammonia and urea in the liver of fed or starved rats. The increase in urea was accompanied by increases in acetylglutamate, ornithine and citrulline. In perfused liver, the addition of an ammonium salt to the perfusate caused increases in acetyl-glutamate and citrulline as observed in vivo, while the concentration of ornithine, one-fifth of that in vivo, did not respond to the addition. From these results, we conclude that ammonia, a direct substrate for urea synthesis, may control the concentrations of both ornithine and acetylglutamate in the liver, directly or indirectly, although perfused liver lacks some factors that keep the concentration of ornithine at the in vivo level and cause its increase in response to ammonia. In another part of the work, enzymological analysis in the liver of 4 cases of so-called adult-type citrullinemia with neuro-psychiatric symptoms of late-onset (24–48 years old) caused by ASS abnormalities was performed. One patient in whose family there was a hereditary tendency, judging from their serum citrulline levels, had an abnormal ASS with very high Km values for citrulline and aspartate. This was similar to the cases of neonatal-type citrullinemia summarized by Shih (13). We also observed another type of citrullinemia: decreased ASS activity in the liver of three patients was associated with the decrease in the enzyme protein. ASS activity in the kidney, however, was not decreased at all. The kinetic properties of the enzymes of the patients were quite similar to those of healthy control. No hereditary tendency was observed in these families. Thus, we assume that adult-type citrullinemia results from an epigenetic abnormality as well as a genetic one and heterogeneity in the cause may explain the higher incidence of citrullinemia in Japan.

Effect of cyclic polylactates on tumor cells and tumor bearing mice

We studied the effect of cyclic polylactates ranging in size from a degree of polymerization number of 3 to 13 on pyruvate kinase, lactic dehydrogenase, anaerobic glycolysis, growth of tumor cells and survival of tumor bearing mice. Pyruvate kinase and lactic dehydrogenase activities were both inhibited by cyclic polylactates, and the inhibition mechanism of cyclic polylactates on lactic dehydrogenase was noncompetitive. About half the anaerobic glycolytic activity of FM3A ascites tumor cells was inhibited and tumor cell growth was also effectively inhibited by cyclic polylactates. Mice, which were treated with cyclic polylactates after inoculation of FM3A ascites tumor cells lived significantly longer than mice, which were treated with vehicle or non mice.

Enhancement by α-1-antichymotrypsin of antibody response in vivo

Summary The immunological effect of α-1-antichymotrypsin on the antibody response in vivo was studied in mice. The plaque-forming cell response to sheep erythrocytes was enhanced by an intravenous injection of α-1-antichymotrypsin. α-1-antichymotrypsin was effective if injected 2 days before or simultaneously with the injection of sheep erythrocytes. Immunoadsorption with an anti α-1-antichymotrypsin-Sepharose column indicated that α-1-antichymotrypsin itself was responsible for enhancement of plaque-forming cell response.

Penetration of Lysophosphatidylcholine into the Dermis

The ability of Lysophosphatidylcholine (LPC) to penetrate into the dermis and its degradation were investigated in vivo using hairless rats. Phosphatidylcholine (PC) was used as a control. Radioactively labelled LPC and PC were applied on the surface of skin, and the radioactivities of the epidermis and dermis were measured at 8, 24, and 48 hours. The recoveries of radioactive materials of LPC and PC within the area of epidermis and dermis at 8 hours were 0.21% and 0.25%; at 24 hours, 0.68% and 0.31%; and at 48 hours, 0.42% and 0.92%. No radioactivity was detected in serum. The radioactive substances which had penetrated were identified as LPC, PC, diglycerides, and free fatty acids. It was also found that topical application of LPC did not change the structure of skin as seen by microscopical examination.

Alteration of DNA primase activity by phosphorylation and de-phosphorylation of histone H1

To investigate the effect of histone H1 on DNA primase activity, partially purified DNA primase from mouse FM3A cells was used. It was found that histone H1 dose dependently inhibited DNA primase. Interestingly phosphorylation of histone H1 reduced the inhibitory activity of the histone. However, de-phosphorylation of the phosphorylated histone H1 resumed the inhibitory activity of DNA primase. These findings lead us to the assumption that phosphorylation and de-phosphorylation of histone may regulate the cell cycle by controlling DNA synthesis through reverse inhibition of DNA primase.