Purification, molecular cloning, and sequencing of phospholipase C-beta 4

Abstract

We have characterized inositol phospholipid-specific phospholipase C (PLC) isozymes in bovine retina. Chromatography of a retinal homogenate on a heparin column partially resolved six peaks of PLC activity, which differed in their relative selectivities for the substrates phosphatidyl 4,5-bisphosphate (PIP2) and phosphatidylinositol (PI). Five of the peaks were shown to correspond to the known PLC isozymes PLC-beta 1, PLC-beta 3, PLC-gamma 1, PLC-delta 1, and PLC-delta 2. PLC-beta 1, PLC-beta 3, PLC-gamma 1, and PLC-delta 1 in the retinal fractions were identified by immunoblotting with isozyme-specific antibodies, and PLC-delta 2 was identified by direct sequencing of tryptic peptides. PLC-gamma 2 and PLC-beta 2 were not detectable by immunoblot analysis. In addition to five of the seven mammalian PLC isozymes identified to date, bovine retina contained a previously unidentified PLC, which exhibited the highest selectivity for PIP2 over PI. The new PLC was purified from a retinal particulate fraction to yield a preparation that contained a major protein band with an apparent molecular mass of 130 kDa on SDS-polyacrylamide gels. Sequence analysis of 12 tryptic peptides derived from the 130-kDa protein suggested that the primary structure of the new PLC is similar to those members of beta-type PLC isozymes, especially to that of PLC-norpA, which was originally identified in Drosophila eye. The new enzyme was thus named PLC-beta 4. A search of a rat brain cDNA library with the polymerase chain reaction and oligonucleotide primers based on common PLC amino acid sequences resulted in the cloning of a rat brain cDNA corresponding to a previously uncharacterized PLC. The cDNA encoded a putative polypeptide of 1176 amino acids, with a calculated molecular mass of 134,532 daltons, that contained the sequences of all 12 tryptic peptides of PLC-beta 4. Furthermore, the deduced amino acid sequence of the encoded protein was more related to PLC-norpA than to any of the three mammalian PLC-beta isozymes. These results suggest that the brain cDNA encodes PLC-beta 4, which is likely a mammalian homolog of PLC-norpA.