Purification of a novel phospholipase C isozyme from bovine cerebellum.
An isozyme of phosphoinositide-specific phospholipase C (PLC) was purified to near homogeneity from bovine cerebellum by a combination of several column chromatography procedures. Approximately 80 micrograms of pure enzyme were obtained from 4 kg of bovine cerebellum, with a final specific activity of 7.5 mumol/min/mg protein in the presence of 0.1% deoxycholate. The enzyme is specific for phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but does not hydrolyze phosphatidylcholine. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis is approximately 97,000. Polyclonal antibodies to previously characterized PLC isozymes, PLC-beta 1, -beta 2, -gamma 1, -gamma 2, and - delta 1, did not cross-react with the purified cerebellar enzyme. Moreover, polyclonal antibodies prepared against the cerebellar enzyme did not react with purified PLC-beta 1, -beta 2, -gamma 1, -gamma 2, or -delta 1. However, the cerebellar enzyme was recognized by two antibodies generated against peptide sequences common to mammalian PLC isozymes. Comparison of partial amino acid sequences of the purified cerebellar enzyme with the deduced amino acid sequence of each known PLC isozyme shows that the cerebellar enzyme is a novel PLC, which could be classified as a PLC-beta-type isozyme. Thus, we have designated this enzyme PLC-beta 4.
Min
D S
DS
Department of Life Science, Pohang Institute of Science and Technology, Hyoja-Dong, Korea.
Kim
D M
DM
Lee
Y H
YH
Seo
J
J
Suh
P G
PG
Ryu
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002531:Cerebellum / Q000201:enzymology*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000235:genetics
D007527:Isoenzymes; D010446:Peptide Fragments; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D010450:Endopeptidases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium