Phosphatidylinositol 4,5-bisphosphate phospholipase C and phosphomonoesterase in Dunaliella salina membranes

In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2). Based on release of [3H]inositol phosphates, the plasma membrane exhibited a PIP2-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast 'bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [3H]inositol phosphates released were recovered as [3H]inositol trisphosphate (IP3). These results suggest a plausible mechanism for the rapid breakdown of PIP2 and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [3H]inositol phospholipids during these assays revealed that some of the [3H]-PIP2 was converted to [3H]phosphatidylinositol 4-monophosphate (PIP) and to [3H]phosphatidylinositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP2/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free [Ca2+]. We also report here that 100 micromolar GTP gamma S stimulates phosphospholipase C activity over a range of free [Ca2+]. Together, these results provide evidence that the plasma membrane PIP2-phospholipase C of D. salina may be subject to Ca2+ and G-protein regulation