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 mumol/min/mg of protein with 1 microM Ca2+. 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 beta isozymes. Reconstitution of purified squid PLC with an AlF(-)-activated 44-kDa G protein alpha subunit extracted from squid photoreceptor membranes resulted in a significant increase in PIP2 hydrolysis over a range of Ca2+ concentrations while reconstitution with mammalian Gt alpha or Gi 1 alpha was without effect. These results suggest that cephalopod phototransduction is mediated by G alpha-44 activation of a 140-kDa cytosolic PLC.
Mitchell
J
J
Department of Pharmacology, University of Toronto, Canada.
Gutierrez
J
J
Northup
J K
JK
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D049832:Decapodiformes; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017956:Photoreceptor Cells, Invertebrate / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium