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, Gq, which stimulates phospholipase C (PLC) activity. We have previously purified a 140-kDa PLC enzyme from squid photoreceptors that is regulated by squid Gq. 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 microM Ca2+ with a specific activity of 10.3 micromol 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 GTPgammaS-bound soluble squid Gq 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 microM. These results suggest that cephalopod phototransduction is mediated by Gq activation of more than one cytosolic PLC enzyme.
Mitchell
J
J
Department of Pharmacology, University of Toronto, Ontario, Canada. jane.mitchell@utoronto.ca
Mayeenuddin
L H
LH
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D049832:Decapodiformes; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D017956:Photoreceptor Cells, Invertebrate / Q000201:enzymology*; D014357:Trypsin; D010738:Type C Phospholipases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D010738:Type C Phospholipases; D014357:Trypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D019204:GTP-Binding Proteins; D002118:Calcium