Purification and characterization of PLC-beta m, a muscarinic cholinergic regulated phospholipase C from rabbit brain membrane.
Two isozymes of phosphoinositide-specific phospholipase C were isolated and purified from salt-washed rabbit brain membranes. The membranes were extensively washed with isotonic, hypertonic and hypotonic buffers prior to solubilization with sodium cholate. Two isozymes (PLC-IV and PLC-beta m) were purified by a combination of DEAE-Sephacel, AH-Sepharose, heparin-Sepharose, AcA-34 gel filtration and mono-Q FPLC chromatographies. The major activity (PLC-beta m) was purified to homogeneity and had an estimated molecular weight of 155,000 on sodium-dodecyl sulfate-polyacrylamide gels (SDS-PAGE). This isozyme was immunologically identified as PLC-beta, an isozyme previously characterized in bovine brain cytosol and 2 M KCl membrane extracts. A second isozyme, PLC-IV, was immunologically distinct from PLC-beta and PLC-gamma and was purified to a stage where three protein bands (Mr 66,000, 61,000 and 54,000) on SDS-PAGE correlated with enzyme activity. The catalytic properties of the isozymes were studied and found to be very similar. The specific activities for PIP2 were greater than those obtained when PI was used. Both PLC-IV and PLC-beta m were Ca2(+)-dependent; near maximal stimulation for PI and PIP2 hydrolysis was observed at 0.5 microM free Ca2+. Sodium pyrophosphate and sodium fluoride stimulated phospholipase C activity of both isozymes. Polyclonal antibodies raised against PLC-beta m were able to inhibit carbachol and GTP gamma S stimulated phospholipase C activity in 2 M KCl washed rabbit cortical membranes. This suggests that in rabbit brain muscarinic cholinergic stimulation regulates PLC-beta m.
Carter
H R
HR
Department of Biochemistry, University of Tennessee, Memphis 38163.
Wallace
M A
MA
Fain
J N
JN
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography / Q000379:methods; D007527:Isoenzymes / Q000302:isolation & purification*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D011817:Rabbits; D011976:Receptors, Muscarinic / Q000378:metabolism*; D010738:Type C Phospholipases / Q000302:isolation & purification*
D007527:Isoenzymes; D011976:Receptors, Muscarinic; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase