The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.
N-Acetyl-L-glutamate synthetase was purified approximately 300-fold from the extract of rat liver mitochondria. The enzyme shows a high substrate specificity for L-glutamate and acetyl-CoA. No or very low activity is observed with other amino acids and acyl compounds. The reaction velocity fits normal Michaelis-Menten kinetics with respect to both L-glutamate (K,, 3.0 mM) and acetylCoA (K,, 0.7 mM).Acetylglutamate, the reaction product, and some of its structural analogues, such as N-acetylaminoadipate, N-propionylglutamate, and N-acetylglutamine, significantly inhibited the enzyme reaction. The Ki for acetylglutamate was 0.07 mM. The enzyme activity is sensitive to thiol-blocking agents and is inhibited by divalent cations.The enzyme activity is markedly stimulated by arginine, presumably by an allosteric mechanism. Other intermediates of the urea cycle and various structural analogues of arginine were practically not effective. Arginine increases the maximal velocity with no influence on K, values for the substrates. The activation shows a sigmoidal dependence on arginine concentrations and reaches a halfmaximal level at 5 -10 p M~ The enzyme behaves as a mixture of multiple forms upon gel filtration (the range of molecular weight, 30000 to 300000) and its mean value is considerably increased in the presence of arginine.The specific activity of the enzyme increases with higher enzyme concentrations in the assay. The effect is more marked in the presence of arginine than in its absence. These observations suggest that the enzyme undergoes association and dissociation with concomitant changes in catalytic properties.Acetylglutamate serves as a specific and obligatory activator of mitochondria1 carbamoyl-phosphate synthetase I, the first enzyme of urea biosynthesis in ureotelic animals including mammals [l, 21. However, the physiological significance of the activator function of acetylglutamate remains obscure. We reported observations [3,4] which support the proposal that varying intracellular levels of acetylglutamate control the activity of carbamoyl-phosphate synthetase I, thus effecting regulation of entry of NH, into the urea cycle [5]. In line with these studies, we demonstrated enzyme activity in mitochondria of rat and mouse livers which catalyzes the acetyl-Co A-dependent acetylation of the amino group of glutamic acid [4]. A unique property of the enzyme is that it is activated specifically by L-arginine.Ahhreoicrtion. Acetylglutarnate, N-acetyl-r-glutamate. Enzymes. Acetylglutamate synthetase or acetyl-CoA: L-glutamate N-acetyltransferase (EC 2.3.1.1); carbamoyl-phosphate synthetase 1 orcarbamoyl-phosphate synthetase (ammonia) (EC 2.7.2.5). This paper deals with partial purification of acetylglutamate synthetase from rat liver and characterization of its kinetic and regulatory properties. A preliminary account of part of this work has already been documented [6]. MATERIALS A N D METHODS Chemicals, Enzymes, and Isotopes
The level of N-acetyl-L-glutamate showed a diurnal rhythm, which was associated primarily with dietary ingestion. Since the hepatic level of arginine, activator for acetylglutamate, is one of the plausible principal factors responsible for the regulation of acetylglutamate level, the effect of arginine was further studied. The intraperitoneal administration of a large amount of arginine increased the level of acetylglutamate and the effect was augmented by the combined administration of glutamine. The arginine administration stimulated the hepatic synthesis of acetylglutamate as measured with [14C]glutamate as tracer, but did not affect the half-life (about 20 min) of the synthesized 14C-labeled acetylglutamate.The effect of arginine in raising the acetylglutamate level was also demonstrated in vitvo in isolated rat liver cells. On the other hand, pent-4-enoic acid, known to inhibit fatty acid oxidation and to decrease acetyl-CoA production in the liver, lowered the level of acetylglutamate in the rat liver cells. It was shown that the cellular ureogenic capacity, as measured with NH4Cl as nitrogen source, showed a close correlation with the varying levels of acetylglutamate, brought about by arginine or pent-4-enoic acid.The carbamoyl phosphate level, which may reflect the balance between its synthesis and utilization for citrulline synthesis, was low (about 1 nmol/g) and remained relatively constant in the livers of intact mice even when the rate of urea synthesis was expected to vary with changes in the dietary protein content. A transient increase in the level was brought about by agents expected to increase the synthesis or to inhibit the utilization, such as ammonia, N-acetylglutamate diethyl ester, or norvaline. These results, together with others, suggest that in intact animals the production of carbamoyl phosphate is coupled to its utilization for citrulline synthesis.
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