Renal glutamic acid decarboxylase (GAD): Solubility and effects of ATP, phosphate and chloride

Glutamic acid decarboxylase (GAD) from the rat renal cortex cannot be solubilized by detergents in contrast to pancreatic and brain GAD. After centrifugation of freshly prepared homogenate, 12% of the initial GAD activity was present in the supernatant and 29% in the sediment. The supernatant and sediment, when incubated together, had 75% of the homogenate activity. GAD was partially solubilized when the homogenate was stored for 24 h at 4 degrees C, and the presence of Triton X-100 increased the solubility. ATP and Pi (2 mmol.l(-1) and 5 mmol.l(-1), respectively) activated GAD in the homogenate. The activation by ATP and Pi was not additive. Solubilized GAD from the homogenate, stored in the absence of Triton X-100 for 24 h at 4 degrees C, was activated by Pi but inhibited by ATP similarly to brain GAD. The inhibition by ATP was specific, other nucleotides showed no inhibitory effect. On the other hand, GAD solubilized in the presence of Triton X-100 was activated by Pi, ATP and other nucleotides. Renal GAD was also activated by chlorides, however sulphates, nitrates and their cations did not influence activity. ATP and chlorides or Pi and chlorides activated it with a synergic effect. The results suggest that renal GAD is composed of three parts: a covalently membrane-bound enzyme, a soluble activator separable by centrifugation and different from ATP, Pi and chlorides, and a third part separable by detergent that modifies the effect of ATP. It is assumed that there are two binding sites for ATP on the enzyme of renal GAD.