Purification, Characterization, and Partial Amino Acid Sequence of a G Protein-activated Phospholipase C from Squid Photoreceptors (*)

Invertebrate visual transduction is thought to be initiated by photoactivation of rhodopsin and its subsequent interaction with a guanyl nucleotide-binding protein (G protein). The identities of the G protein and its target effector have remained elusive, although evidence suggests the involvement of a phospholipase C (PLC). We have identified a phosphatidylinositol-specific PLC from the cytosol of squid retina. The enzyme was purified to near-homogeneity by a combination of carboxymethyl-Sepharose and heparin-Sepharose chromatography. The purified PLC, identified as an approximately 140-kDa protein by sodium dodecyl sulfate-polyacrylamide gels, hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2) at a rate of 10-15 μmol/min/mg of protein with 1 μM Ca. The partial amino acid sequence of the protein showed homology with a PLC cloned from a Drosophila head library (PLC21) and lesser homology with Drosophila norpA protein and mammalian PLC β isozymes. Reconstitution of purified squid PLC with an AlF-activated 44-kDa G protein α subunit extracted from squid photoreceptor membranes resulted in a significant increase in PIP2 hydrolysis over a range of Ca concentrations while reconstitution with mammalian Gtα or Gi1α was without effect. These results suggest that cephalopod phototransduction is mediated by Gα-44 activation of a 140-kDa cytosolic PLC.

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