The physicochemical, catalytic, and immunochemical properties of rat liver argininosuccinate synthetase [EC 6. 3. 4. 5] purified and crystallized as reported earlier (Saheki et al. (1975) FEBS Lett. 58, 314-317) are described in the present paper.
1. The molecular weight of the enzyme was estimated to be 158, 000 by Sephadex G-200 get filtration, 207, 000 by sedimentation equilibrium centrifugation, and 192, 000 on the basis of the molecular weight of the subunit and of the quaternary structure of the enzyme.
2. Each of the subunits had the same molecular weight of 48, 000, as reported earlier, and showed identical electrophoretic mobility on an acrylamide gel with 6 M urea. No free terminal amino group was detectable on dansylation followed by thin layer chromatography.
3. The enzyme was rich in glutamic acid, lysine, aspartic acid, valine, leucine, and glycine. The isoelectric point was found to be pH 7.9 by electrofocusing.
4. Sixteen residues of half-cystine were found, and all of them could be titrated by 5, 5'-dithio-bis (2-nitrobenzoic acid) as free sulfhydryl groups in the presence of sodium dodecyl sulfate. Only one sulfhydryl group per subunit was titrated in the absence of sodium dodecyl sulfate, and its blockage caused inhibition of the enzyme activity by up to 80%.
5. Catalytic constants such as Km values and pH optimum of the purified rat liver argininosuc-cinate synthetase were quite similar to those of the partially purified steer liver and hog kidney enzymes reported by Rochovansky and Ratner ((1967) J. Biol. Chem. 242, 3839-3847).
6. Among the various nucleotide triphosphates tested, only ATP served as a substrate for the enzyme; the others slightly inhibited the reaction.
7. In Ouchterlony double-diffusion analysis using antisera to the purified enzyme, both the purified and the crude preparations gave a single band with complete fusion. The enzyme was inhibited by up to 85% on addition of the antiserum.