Abstract
The effect of guanine nucleotides on platelet and calf brain cytosolic phospholipase C was examined in the absence of membranes or detergents in an assay using labeled lipid vesicles. Guanine nucleotides stimulate hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PtdIns-4,5-P2) catalyzed both by enzyme from human platelets and by partially purified enzyme from calf brain. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was the most potent guanine nucleotide with a half-maximal stimulation at 1-10 microM, followed by guanosine 5'-(beta, gamma-imido)triphosphate greater than GTP greater than GDP = guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(2-thiodiphosphate) was able to reverse the GTP gamma S-mediated stimulation. NaF also stimulated phospholipase C activity, further implying a role for a guanine nucleotide-binding protein. In the presence of GTP gamma S, the enzyme cleaved PtdIns-4,5-P2 at higher pH values, and the need for calcium ions was reduced 100-fold. The stimulation of PtdIns-4,5-P2 hydrolysis by GTP gamma S ranged from 2 to 25-fold under various conditions, whereas hydrolysis of [3H]phosphatidylinositol was only slightly affected by guanine nucleotides. We propose that a soluble guanine nucleotide-dependent protein activates phospholipase C to hydrolyze its initial substrate in the sequence of phosphoinositide-derived messenger generation.
Highlights
From the Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine, St
Our experiments were designed to test thehypothesis that the soluble phospholipase C enzyme in platelets and brain might be stimulated by a guanine nucleotide-binding protein and be similar to the membrane-bound polyphosphoinositide-specific phospholipase C activity
The lipids were tracer-labeled with tritiated phosphoinositides, which did not change the lipid composition of the vesicles since the added labeled lipids were never more than 1%of the mass of the endogenous phosphoinositides (18 nmol of PtdIns, 1.4 nmol of PtdIns-4,5-P2/1O9 platelets [32])
Summary
From the Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine, St. To detect an effect of pertussis toxin on the phospholipase C activation, platelet supernatant (100 pg of protein) wasmixed with either 2 or 15 pg of preactivated A protomer of pertussis toxin, in the presence of 1 mM NAD in i;Imphate buffer, in a total volume of 100 pl for 60 min a t 30 “C. 50 p1 (100 pg of protein) of Activity was measured as a function of time using 8 (A) or 24 (0)pg this, or 20 pg of partially purified brain phospholipase C, or platelet of platelet supernatant protein in the absence (closedsymbols) or membranes (45pg ofprotein) was mixedwith 10pl of 0.1 M potassium presence (open symbols) of 100 p~ GTPyS at pH 6.0. Other Methods-Protein was measured using the Bio-Rad protein assay with bovine serum albumin as a standard
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