Abstract
A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.
Highlights
Stores beforebeing rapidly turned over through a complex metabolic pathway to inositol [3]
Myelin basic protein (MBP) stimulated the PI-PLC described in mammalian brain
We report the isolation of a 57-kDa PI-PLC from bovine brain that has an amino acid composition similar to the 65-kDa PI-PLC from sheep seminal vesicles reported by Hofmann and Majerus [7]
Summary
L-a-1-Stearoyl-2-arachidonoyl [arachidonoyl-l-‘“Clphosphatidylcholine (specific activity, 60 mCi/mmol), L-a-1-palmitoyl-2-arachidonoyl [arachidonoyl-1-“Clphosphatidylethanolamine (specific activity, 60 mCi/mmol), L-cu-l-stearoyl-2-arachidonoyl [arachidonoyl-l-. To prepare the enzyme balance sheet, approximately 8 x lo dpm of [2-3H]PI (specific activity, 200 dpm/nmol) was incubated for 60 min as mentioned above. These conditions were used to determine the enzyme specificity and to generate substrate kinetic curves, Purification Procedure. "CIPI, [ino~itol-2-~H]PoIr, [inosit01-2-~H]PIP2were incubated sep- Other solutions such as that used for blocking, MBP of different arately for 60 min at 37 "C with or without enzyme in the presence concentrations, and primary and secondary antibodies were the same of 4 mM Ca2' except when PIP2 was used in which case the Ca" as described for the ELISA. The proteins that eluted in the void volume were dialyzed against water and lyophilized
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