Abstract
Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.
Highlights
Two peaks of phosphoinositide-specific phospholi- A variety of hormones, neurotransmitters, andgrowth facpase C (PI-phospholipase C (PLC)) activity were resolved when guinea tors induce the rapid catabolism of phosphoinositides via a pig uterus cytosolicproteins werechromatographed on receptor-mediated process
Cells were stimulated with 1 PM PMA and at the indicated time, total proteins were extracted and separated hy two-dimensional gel electrophoresis as described under "Materials and Methods." The spot corresponding to PI-PLC I was excised from the gel and the radioactivity determined by liquid scintillation spectroscopy
Form I was purified to apparenthomogeneity as determined by SDS-polyacrylamide gel electrophoresis, and is similar inchemical and kinetic propertiesto PI-PLCI from sheep seminal vesicle [31]
Summary
Agonist-induced phosphatidylinositol hydrolysis is mediated by a phosto was calcium-dependent, whereas significant hy- phoinositide-specific phospholipase C (PLC). PLC I from guinea pig uterus exhibited similar pH optima and kinetic parameters as sheep semivneaslicle PLC [31].As previously reported[31], deoxycholatemarkedly enhanced enzyme activity (data not shown); all subsequent. The substrate preference and calcium requirements of PI-PLC I isolated from guinea pig uterus cytosol are consistentwith this enzyme being coupled to agonist-induced phosphoinositide turnover. Antibodies generated against PI-PLC Ib reacted with a 62-kDa protein in cytosolic and membrane fractions from guinea pig uterus (Fig. 10B). These data demonstrate that PI-PLC I is not a proteolytic product of a higher molecular weight protein. The phospholipase C activity in eachfraction was determined using 10 p~ phosphatidylinositol 4,5-bisphosphate as a substrate as described under “Materials and Methods.”
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