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

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 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.

Identification and Functional Characterization of the Moss Physcomitrella patens Δ5-Desaturase Gene Involved in Arachidonic and Eicosapentaenoic Acid Biosynthesis

“…On the other hand, in yeast a similar cytochrome b 5 domain was recognized in the ⌬ 9 -acyl-CoA desaturase (OLE1), but in this enzyme it was located at the C terminus. However, when the cytochrome b 5 region was deleted from this gene, the yeast cells remained fatty acid auxotrophs, even in the presence of the endogenous yeast cytochrome b 5 (33,42). This suggests that the cytochrome b 5 domain plays an essential role in the desaturation reaction.…”

“…3) and the presence of a heme binding region characterized by the HPGG motif toward the N terminus (32). A heme-containing electron donor is required for fatty acid desaturation, and cytochrome b 5 fulfills this function for the membrane-bound desaturase (16,33,34). Therefore, the whole sequence can be considered as coding for a fusion protein consisting of an N-terminal cytochrome b 5 and a desaturase.…”

A β‐subclass phosphatidylinositol‐specific phospholipase C from squid (Loligo forbesi) photoreceptors exhibiting a truncated C‐terminus

“…Two regions in the C-terminus offll-PLC recently defined (designated 'P box' found to be responsible for association with the particulate fractions, and 'G box' containing sequences involved in interaction with G~q) [43] appear not to be represented in the truncated squid PLC post 'Y' region. A cytosolic PLC was recently purified from the retina of an undefined species of squid (44). This PLC ran as a 140 kDa species on SDS-PAGE and was activated by G protein ~ subunit.…”

Squid visual arrestin: cDNA cloning and calcium‐dependent phosphorylation by rhodopsin kinase (SQRK)

“…The resulting salt-washed (SW) membranes were centrifuged at 100 000 g for 30 min at 4°C. Salt-washed membranes were resuspended and Dounce-homogenized in 90 mL 34% sucrose (w/w) in solution A and rhabdomeric membranes prepared as previously described (Mitchell et al 1995). These washed rhabdomeric membranes were free of rhodopsin kinase as assessed by immunoblotting using the SQRK antibody.…”

“…Photoreceptor membranes were prepared from frozen eyes taken from freshly caught Loligo pealei as previously described (Mitchell et al 1995). Briefly, 50 frozen squid eyes were thawed in 100 mL of buffer A containing 10 mM Hepes pH 7.5, 2 mM EGTA, 3 mM MgSO 4 , and 100 lM AEBSF, and vortexed to prepare crude homogenate.…”