Purification of N-acetyl-L-glutamate synthetase from rat liver mitochondria and substrate and activator specificity of the enzyme

Abstract

N-Acetylglutamate synthetase was purified 33,000-fold to apparent homogeneity from the matrix fraction of rat liver mitochondria. The purification procedure involved treatment of mitochondria with digitonin, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, Affi-Gel blue chromatography, acetylglutaminyl Bio-Gel chromatography, sucrose density gradient centrifugation, and isoelectric focusing. As the enzyme lost activity in contact with a glass surface, glassware coated with silicone was used throughout the purification. Triton X-100 stabilized the enzyme and was included in most buffers at a concentration of 0.1% (w/v). The purified acetylglutamate synthetase has a specific activity of 92.4 mumol min-1 (mg of protein)-1, in the presence of 1 mM L-arginine. The enzyme had a sedimentation coefficient (S20,w) of 8.1 S and its molecular weight was estimated to be about 160,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 57,000, indicating a composition of three subunits of similar size. The purified acetylglutamate synthetase showed a high substrate specificity for L-glutamate and acetyl-CoA, confirming the previous results with a less purified preparation (Shigesada, K., and Tatibana, M. (1978) Eur. J. Biochem. 84, 285-291). Among other 14 acyl-CoAs tested, only propionyl-CoA could substitute for acetyl-CoA, as an acyl donor, with a reaction rate 4.3% of that with acetyl-CoA at 0.5 mM. Among amino acids other than L-glutamate and derivatives or analogues of glutamate tested, the following served as an acyl acceptor: glycine at a rate 2.7% of that with L-glutamate at 1.0 mM, L-glutamine (5.0%), L-glutamate gamma-hydroxamate (15.5%), DL-alpha-aminoadipate (5.2%), and DL-alpha-aminopimelate (4.0%). The purified enzyme is markedly stimulated by L-arginine. The specificity of L-arginine as a low molecular weight activator was strict; only L-argininic acid could activate the enzyme to a lesser extent. Cationic polypeptides, such as protamine, polyarginine and polylysine, also activated the synthetase. The effect was additive to that of arginine.