Determination of Strength and Specificity of Membrane-Bound G Protein-Phospholipase C Association Using Fluorescence Spectroscopy

Two kinds of informations about arachidonic acid (AA) metabolism in platelet phospholipids (PL) have been obtained from the use of purified phospholipases: 1) Beside the determination of PL sidedness in the plasma membrane, non-lytic degradation by phospholipase A2 + sphingomyelinase C showed that only 6 % of the total platelet AA is localized in the outer surface of the plasma membrane. This heterogeneous distribution is actually a consequence of PL asymmetry, since sphingomyelin and phosphatidylcholine, which predominate in membrane outer leaflet, contain only traces or relatively lower amounts, respectively, of AA than the internal lipids. It is further shown that incubating platelets with free AA specifically labels the large internal pool of AA, whereas the small external pool is renewed by a direct exchange of phosphatidylcholine with plasma lipoproteins. This offers a doublelabelling method allowing to explore the exact role of each AA pool.2) Platelet aggregation by Clostridium welchii phospholipase C produces the same metabolic changes (accumulation of phosphatidic and lysophosphatidic acids) as those induced by thrombin. These observations have led to describe the existence of a cytosolic phosphatidylinositol-specific phospholipase C and a membrane-bound diglyceride lipase. Both enzymes, coupled to diglyceride− (and monoglyceride−) kinase(s), could achieve AA release and (lyso) phosphatidic acid accumulation. Some properties of these enzymes (subcellular localization, calcium and pH dependence, positional specificity) will be presented.

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Membrane-binding properties of phospholipase C-β1 and phospholipaseC-β2: role of the C-terminus and effects of polyphosphoinositides, G-proteins and Ca2+

Biochemical Journal ◽

10.1042/bj3270431 ◽

1997 ◽

Vol 327 (2) ◽

pp. 431-437 ◽

Cited By ~ 22

Author(s):

M. John JENCO ◽

P. Kevin BECKER ◽

J. Andrew MORRIS

Keyword(s):

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Membrane Binding ◽

Molar Ratio ◽

Phospholipid Vesicles ◽

C Terminus ◽

Catalytically Active ◽

Membrane Bound ◽

Active Fragments

We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-β1 and -β2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10-3 M. PLC-β1 and PLC-β2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10-5 M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-β1 and PLC-β2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein α- and βγ-subunit activators in the phospholipid vesicles does not increase the binding of PLC-β1 or PLC-β2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10-6 M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10-3 M). PLC-β1 and -β2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.

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