Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney.
A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.
D000818:Animals; D003980:Diazooxonorleucine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005971:Glutamates / Q000494:pharmacology*; D018698:Glutamic Acid; D005972:Glutaminase / Q000037:antagonists & inhibitors*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D007982:Levulinic Acids / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D010710:Phosphates / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D005971:Glutamates; D007982:Levulinic Acids; D010710:Phosphates; D003980:Diazooxonorleucine; D018698:Glutamic Acid; C021959:2-amino-4-oxo-5-chloropentanoate; D005972:Glutaminase