Glutaminase from Pig Renal Cortex

A method for preparing highly purified glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) from pig renal cortex is described. The main steps consist of sodium sulfate fractionation followed by alternative solubilization by dialysis against Tris-HCI buffer and precipitation with phosphateborate. The linal enzyme preparation contains no impurities which could be detected by sucrose gradient centrifugations, sedimentation velocity experiments, immune diffusion, or disc electrophoresis. Glutaminase has been found to exist in different interconvertible molecular forms. A Tris-soluble enzyme form (Tris-HCl enzyme), obtained by dialysis against Tris-HCl, has an average sedimentation coefficient of 7.3 and an apparent molecular weight of 140,000 to 160,000, as determined by sucrose gradient centrifugation and Sephadex gel filtration. Another enzyme form (phosphate enzyme), obtained by dialysis of the Tris-HCl enzyme against phosphate, has an average sedimentation coefficient of 10.8 and an apparent molecular weight of 250,000 to 290,000. A phosphate-borate-precipitated form (phosphate-borate enzyme), with an average sedimentation coefficient of 50 and an apparent molecular weight of about Z,OOO,OOO, is obtained by addition of borate to the phosphate enzyme. Sulfate has an effect on the sedimentation coefficient similar to that of phosphate, and glutamate, citrate, and malonate have an effect similar to that of Tris-HCI. The sedimentation coefficients of both the Tris-HCl enzyme and the phosphate enzyme are increased by borate and the dye bromthymol blue. The sedimentation coefficient of the phosphate enzyme is lowered by chloride and urea. The Tris-HCl enzyme has a pH optimum of about pH 9, whereas no distinct optimum in the pH range 8 to 9 has been shown for the phosphate-borate enzyme. These forms of glutaminase are activated not only by anions, but also by ammonium ions and bromthymol blue, which suggests allosteric properties.