Some Properties of Argininosuccinate Synthetase Purified from Human Liver and a Comparison with the Rat Liver Enzyme

Abstract

In an attempt to clarify the pathogenesis of citrullinemia, which is caused by argi. ninosuccinate synthetase (ASS) [EC 6. 3. 4. 5] deficiency and is observed in extraordinarily high incidence in Japan, some properties of ASS purified to homogeneity from human liver were compared with those of ASS purified from rat liver.
The molecular weights of the subunit and native form of purified human liver ASS were estimated to be 45, 000 and 185, 000, respectively, which are the same as those of rat liver ASS. Human liver ASS migrated as a single band on polyacrylamide gel electrophoresis at pH 8.9 but showed multiple bands at pH 6.6 and on isoelectrofocusing in thin layer polyacrylamide gel, as did rat liver ASS. There was almost no difference in the apparent K_m values for three substrates between human and rat liver ASSs. Both enzymes showed negative cooperativity in respect to ATP. Rat liver ASS also showed negative cooperativity in respect to one amino acid substrate with the other amino acid substrate at unsaturated concentrations, as reported for bovine liver ASS, while human liver ASS showed only slight cooperativity effects.
Immunological analyses also showed a high degree of similarity between the enzymes. The amino acid composition of human liver ASS was very similar to that of rat liver ASS. Differences in the amounts of amino acid residues per subunit between human and rat liver ASSs were within 10% except for serine, phenylalanine, and arginine. The peptide patterns on SDS-polyacrylamide gel electrophoresis after digestion of the enzymes by trypsin were almost indistinguishable from each other. These results suggest that the primary structures of the human and rat liver enzymes are very similar.