Characterization of fMet-Leu-Phe-stimulated phospholipase C in streptolysin-O-permeabilised cells.
Phospholipase C (specific for inositol lipids) is known to be present both in membranes and cytosol. Receptor-mediated activation of this enzyme occurs via a guanine nucleotide regulatory protein (G-protein), designated Gp. We have compared the stimulation of this enzyme by fMet-Leu-Phe via the G-protein in HL60 membranes and in permeabilised cells. fMet-Leu-Phe stimulated phospholipase C in membranes at 2 min and the response was dependent on exogenously added GTP. GTP alone also stimulated phospholipase C activity such that at 10 min the response to fMet-Leu-Phe was minimal. In comparison, the response to fMet-Leu-Phe in permeabilised cells was greater in extent but did not require added GTP. However, it was antagonized by GDP analogues (GDP[beta S] greater than GDP greater than dGDP) and by pertussis toxin pretreatment, indicating that fMet-Leu-Phe-stimulated phospholipase C activity was also mediated via Gp. GTP and its analogue GTP[gamma S] also stimulated phospholipase C and their effects were strictly additive to the stimulation obtained with fMet-Leu-Phe. Such additivity was also observed when two receptor-directed agonists, fMet-Leu-Phe and ATP, were used to stimulate intact cells. It is concluded that (a) the size of the response with fMet-Leu-Phe in membranes is limited by the loss of a component, possibly phospholipase C, and (b) stoichiometry and physical organisation of multiple species of G-proteins and/or phospholipases C may explain the independent nature of phospholipase C activation by fMet-Leu-Phe, ATP and guanine nucleotides.
Stutchfield
J
J
Department of Physiology, University College London, England.
Cockcroft
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D037342:Pertussis Toxin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism; D013301:Streptolysins / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D001426:Bacterial Proteins; D006150:Guanine Nucleotides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013301:Streptolysins; D010566:Virulence Factors, Bordetella; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D002118:Calcium