Abstract
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.
Highlights
The cerebellar enzywmaes recognized bytwo Phosphoinositide-specificphospholipase C (PLC) isozymes to cell surface receptors [36,37,38,39]
We report purification of a PLCisozyme that appears to be distinct from the mammalian isozymes purified so far
From comparison of the partial amino acid sequence of this isozyme with those of the known PLCs we suggest the new enzyme be designated PLC-04
Summary
The cerebellar enzywmaes recognized bytwo PLC isozymes to cell surface receptors [36,37,38,39]. We have designated this enzyme class, Gal and GaI6,have been reported [48], andcDNAs for PLC-84. HL60 cells and WI-38 fibroblast cells, respectively [27], activation of PLC isozymes by GaI4 andGa16has not yet been. From comparison of the partial amino acid sequence of this isozyme with those of the known PLCs we suggest the new enzyme be designated PLC-04. Comparison of the deduced amino acid sequences of Materink the cloned PLCs suggested thatthere exist at leastthree. Phatidylinositol, bovine brain phosphatidylinositol 4,5-bisphosphate, and formalin-fixed Staphylococcus aureus cells (Pansorbin) were obtained from Calbiochem. PLC-PI, -71, or -61 were purified from bovine brain as described previously [8].PLC-p2 and -72 were obtained from Dr Sue Goo Rhee, National Heart, Lung, and Blood Institute
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