Kinetic analysis of guanosine 5'-O-(3-thiotriphosphate) effects on phosphatidylinositol turnover in NRK cell homogenates

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Abstract
Addition of guanine nucleotide analogue guanosine 5''-O-(3-thiotriphosphate) (GTP.gamma.S) to [3H]inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP-.gamma.S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP.gamma.S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP.gamma.S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP.gamma.S addition (less than 2 min) while inositol 1,3,4-tris(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both [3H]inositol and [32P]phosphate fluxes by nonlinear least-squares analysis. GTP.gamma.S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP.gamma.S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP.gamma.S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.

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