Abstract
Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activites were determined using [ 3H]phosphatidyl inositol bisphosphate ([ 3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130 kD was recognized by an antibody against PLCβ4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gqα was also present in these two fractions. GTPγS stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTPγS. The stimulation by light was still observed when irradiated membrane was incubated at 10°C for 10 min and then mixed with GTPγS. These results suggest that stable metarhodopsin stimulates a PLCβ4/norp A-like enzyme via a G-protein, Gq.
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