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Abstract
The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the purified turkey erythrocyte phospholipase C after reconstitution of the enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of purified enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.
Highlights
From the Department and the SDepartment of Pharmacology, of Biochemistry, the Uniuersity of North CarolinaSchool of Medicine, Chapel Hill, North Carolina 27599
Turkey Erythrocyte Ghosts-Ghosts prepared from turkey erythrocytes labeled to high specific radioactivity with [3H]inositol (average specific radioactivity of PtdIns[4,5]P, = 0.5 Ci/mmol) were used as acceptor membranes for the reconstitution of turkey erythrocyte phospholipase C (PLC) purified as described in the accompanying manuscript [12]
PLC of turkey erythrocyte ghosts [13, 14, 22,23]. In contrast to these reports, under the conditions used for the experiment presented in Table I, there was little measurable stimulation of PLC activity by 10 PM
Summary
The costs of publication of this article were defrayed in part by the payment of page charges. 1,4,5-trisphosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-. L-ninerazineethanesulfonic acid: ADPBS, adenosine 5’-0-(2-thiodiphosphate); GTPrS, guanosine. The preceding paper describes the purification and properties of a PLC from turkey erythrocytes [12]. We describe the regulation of the purified PLC when reconstituted with [3H]inositol-labeled turkey erythrocyte ghosts. The results demonstrate that this isoenzyme of PLC can function as a catalytic component of the machinery used by cell surface receptors and their associated G-proteins to generate inositol lipid-derived second messengers
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