TIME-DEPENDENT INHIBITION OF PHOSPHOLIPASE CP-CATALYZED PHOSPHOINOSITIDE HYDROLYSIS - A COMPARISON OF DIFFERENT ASSAYS

The properties of three different beta-isoforms of phospholipase C (PL C) were analysed using substrate lipids dispersed in phospholipid vesi cles, phospholipid-detergent mixed micelles and phospholipid monolayer s spread at an air-water interface. PhosphatidylinositoI 4,5-bisphosph ate hydrolysis went virtually to completion in monolayers, but inosito l trisphosphate production was curtailed prematurely in vesicular and micellar assays. Assays were linear for less than 2 min with vesicles; the linear portion could be significantly extended in micelles by inc reasing the ratio of micelles to enzyme molecules. However, onset of a second lower rate of substrate hydrolysis always occurred when less t han or equal to 10% of PtdIns(4,5)P-2 had been utilized. This was not due to enzyme inactivation in the micellar interface, determined by ad dition of fresh substrate or fresh enzyme after the slow phase of acti vity had started, nor was it due to overt product inhibition of PLC or apparent entrapment of PLC at the micelle surface. These results are similar to those seen in assays using bacterial PLC and we suggest tha t the biphasic kinetics may be due to product-dependent changes in the presentation of substrate lipid to PLC in lamellar assays, leading to reduced activity.