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

doi:10.1074/jbc.270.2.854

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

From the (1)Department of Pharmacology, University of Toronto, Toronto, M5S 1A8 Canada and the
(2)Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892

Abstract

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 G_tα or G_i1α was without effect. These results suggest that cephalopod phototransduction is mediated by Gα-44 activation of a 140-kDa cytosolic PLC.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

G protein

guanine nucleotide-binding protein

PLC

phospholipase C

PIP₂

phosphatidylinositol 4,5-bisphosphate

PE

phosphatidylethanolamine

PI

phosphatidylinositol

Mops

3-(N-morpholino)propanesulfonic acid

Mes

2-(N-morpholino)ethanesulfonic acid

GTPS

guanosine 5′-3-O-(thio)triphosphate

PAGE

polyacrylamide gel electrophoresis.
↵²J. Mitchell, unpublished data.
- Received December 2, 1993.
- Revision received August 8, 1994.