A novel candidate for receptor-coupled phospholipase C purified from human platelets.
The breakdown of polyphosphoinositides (PPI) but not phosphatidylinositol (PI) has been hypothesized as the primary event following agonist (hormones/growth factors/neurotransmitters) stimulation in a wide variety of systems. This, in turn, predicts the existence of a phospholipase C (PLC) enzyme that shows specificity to PPI. Ideally, this PPI-specific PLC activity should not be absolutely dependent on Ca2+ because of its proposed role in Ca2+ mobilization. I have recently identified two PLC activities that are specific to PPI and have described their resolution from a PLC that acts on all three phosphoinositides (Manne, 1987). In this report, I describe purification to near homogeneity of one of these PLC activities. The enzyme shows maximal activity towards PPI in the presence of physiological Mg2+ concentrations, and does not act on PI under conditions optimal for PPI hydrolysis. However, a weak PI hydrolytic activity, representing about 1/8th to 1/20th of that observed with PPI is detected when 0-100 microM Ca2+ is present in the assay. This weak PI hydrolytic activity is strongly inhibited by mM Ca2+, which is required at mM levels for most of the PLC enzymes described in literature. The size of the native enzyme as determined by gel filtration (high performance liquid chromatography) is 140 kDa. Analysis of the purified enzyme by HPLC on Zorbax GF-250 column showed a single major peak that coincided with the enzyme activity. Under both denaturing and non-denaturing conditions of SDS-polyacrylamide gel electrophoresis, the highly purified enzyme shows two major bands of 38 kDa and 42 kDa, which together represent about 90% of the total stain on the gel.
Manne
V
V
Wistar Institute, Philadelphia, Pennsylvania 19104.
D001792:Blood Platelets / Q000201:enzymology*; D002122:Calcium Chloride / Q000494:pharmacology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000097:blood* / Q000302:isolation & purification; D013379:Substrate Specificity
D004533:Egtazic Acid; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D008274:Magnesium; D002122:Calcium Chloride