Purification, G protein activation, and partial amino acid sequence of a novel phospholipase C from squid photoreceptors

Invertebrate visual signal transduction is initiated by rhodopsin activation of a guanine nucleotide binding protein, G(q), which stimulates phospholipase C (PLC) activity. We have previously purified a 140-kDa PLC enzyme from squid photoreceptors that is regulated by squid G(q). In these studies, an additional PLC enzyme was purified from the cytosol of squid photoreceptors and identified as a 70-kDa protein by SDS-polyacrylamide gel electrophoresis. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PLC-70 was optimal at pH 5 in the presence of 100 mu M Ca2+ with a specific activity of 10.3 mu mol min(-1) mg(-1), A polyclonal antibody raised against purified PLC-70 did not recognize purified PLC-140, and proteolytic digestion of the two purified enzymes with trypsin or Staphylococcus aureaus V8 protease showed distinct patterns of peptide fragments, indicating that PLC-70 is not a fragment of PLC-140. The partial amino acid sequence of the protein showed homology with PLC21 and norpA isozymes cloned from Drosophila, and mammalian PLC beta isozymes. Reconstitution of purified GTP gamma S-bound soluble squid G(q) with PLC-70 resulted in significant enhancement of PIP2 hydrolysis over a range of Ca2+ concentrations and shifted the maximum activation by calcium to 1 mu M. These results suggest that cephalopod phototransduction is mediated by G(q) activation of more than one cytosolic PLC enzyme.