Regulation of renal ammoniagenesis. Purification and characterization of phosphate-dependent glutaminase from rat kidney.

Abstract

Abstract Phosphate-dependent glutaminase can be solubilized from rat kidney mitochondria by treatment with high concentrations of nonionic detergents or by lyophilization in the presence of a borate buffer. Borate promotes a reversible polymerization of this enzyme into a high molecular weight aggregate. Removal of borate to produce the low molecular weight Tris form of glutaminase is associated with a reversible threefold decrease in specific activity. This process makes possible the rapid purification (1000-fold) of this glutaminase by successive gel filtration in each of its molecular weight forms. The purified glutaminase is stable at 3 °C in borate, phosphate buffer for at least 6 months. Activation of this enzyme is not specific and can be achieved by a large number of phosphate-containing compounds including pyrophosphate, nucleotides and sugar phosphates. Nucleotides produced a significantly greater percentage activation of the mitochondrial and borate forms of glutaminase than of the Tris form. In addition, only the former two forms are activated by a large number of carboxylic acids. The specific activities of the borate and Tris forms of glutaminase are also increased by the presence of Triton X-100.