Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein.
Phospholipase D in human neutrophil lysates is activated by GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)), implying the participation of a GTP-binding protein. Reconstitution of GTP gamma S-dependent activity requires protein factors in both the plasma membrane and the cytosol (Olson, S. C., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242). The location of the GTP-binding protein was investigated by preincubating either cytosol or plasma membrane with GTP gamma S, followed by removal of all but tightly bound nucleotide and reconstituting activity with the complementing untreated subcellular fraction. This approach indicated that the GTP-binding protein was membrane-associated. A low magnesium requirement for GTP gamma S prebinding, as well as a failure of aluminum fluoride to activate, suggested a Ras-like small M(r) GTP-binding protein. smg GDP dissociation stimulator, which stimulates the exchange of GDP for GTP on a variety of small GTP-binding proteins, stimulated GTP-dependent phospholipase D activity. Rho GDP dissociation inhibitor, a regulatory protein that binds specifically to and inhibits the functions of Rho family small GTP-binding proteins, inhibited GTP gamma S-dependent activity. Thus, neutrophil phospholipase D is regulated by a membrane-associated small molecular weight GTP-binding protein, likely to be a member of the Rho family.
Bowman
E P
EP
Department of Biochemistry, Emory School of Medicine, Atlanta, Georgia 30322.
Uhlinger
D J
DJ
Lambeth
J D
JD
D000328:Adult; D001483:Base Sequence; D004277:DNA, Single-Stranded; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000145:classification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010739:Phospholipase D / Q000378:metabolism*
D004277:DNA, Single-Stranded; D008565:Membrane Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; D019204:GTP-Binding Proteins