Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells.
Phosphoinositide-specific phospholipase C (PLC) activities have been partially purified from cultured vascular smooth muscle cells and analyzed for substrate specificity, calcium and pH requirements, and molecular weight. The purification procedure involved DEAE-cellulose and heparin-Sepharose chromatographies followed by Mono Q and size exclusion high performance liquid chromatography. This technique resolves multiple peaks of activity using phosphatidylinositol (PI) and PI 4,5-bisphosphate (PIP2) as substrates. The major peak was purified to near homogeneity as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PLC activity in vascular smooth muscle cells can be divided into two types based on their calcium and pH requirements, substrate preferences, and molecular weights. The low molecular weight PLC hydrolyzes both PI and PIP2, has a molecular mass of 58 kDa, requires the most calcium for full activation, and has a PI-pH profile that shifts slightly with calcium concentration. Screening a cDNA library with oligonucleotides directed against several of the known PLCs identified a highly expressed PLC cDNA that is 99% homologous to PLC-alpha, suggesting that this low molecular weight peak in fact corresponds to PLC-alpha. The high molecular mass peak (157 kDa) shows much greater activity against PI than PIP2, is active at lower calcium concentrations, and has a PI-pH optimum of 5.0 regardless of calcium concentration. Each of the PIP2 PLC activities is strongly dependent on the relative levels of calcium and pH in the assay buffer. These observations suggest that vascular smooth muscle contains both a high and low molecular weight PLC whose activities are affected markedly by the changes in calcium and pH accompanying hormonal stimulation of the cell.
Griendling
K K
KK
Department of Medicine, Emory University, Atlanta, Georgia 30322.
Taubman
M B
MB
Akers
M
M
Mendlowitz
M
M
Alexander
R W
RW
D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium