Time-dependent activation and inactivation of pig brain glutaminase

Abstract

Time-dependent changes in the activity of soluble and membrane-bound glutaminase from pig brain were examined. The membrane-bound enzyme was relatively stable to inactivation as a consequence of dilution and did not show time-dependent activation by phosphate or borate ions. In contrast the soluble enzyme lost activity rapidly when diluted but the presence of glutamine protected against this inactivation and the competitive inhibitor albizziin also afforded some protection. Inactivation occurred even more rapidly in the presence of glutamate. The stability of the soluble enzyme depended on the buffer in which it was stored and storage in Tris-HCl buffer pH 7.4 resulted in a loss of its ability to be activated by phosphate and borate ions. The kinetics of the activation of the soluble enzyme in Tris-HCl buffer by borate and phosphate ions showed the order of the reaction with respect to enzyme concentration to be 3.5 and 1.9 respectively with Hill constants of 1.8 for borate and 2.2 for phosphate. Activation of a preparation of the soluble enzyme stored in a borate-containing buffer by phosphate ions gave an order of 1.7 with respect to enzyme concentration and a Hill constant for phosphate of 2.4. These results were consistent with the activation processes involving aggregation of the enzyme and the smaller form of the enzyme in Tris-HCl buffer having at least two binding sites each for phosphate and borate ions. The implications of these time-dependent phenomena for assay of the activity of the soluble enzyme are also discussed.