Characterization of fMet‐Leu‐Phe‐stimulated phospholipase C in streptolysin‐O‐permeabilised cells

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Abstract
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[βS] > GDP > 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[γS] 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.

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