ABSTRACT. The amount of free calcium in the cytoplasm is important in stimulation coupled with a number of cellular functions. The putative iono-

ABSTRACT.Ca2+-and phospholipid-dependent protein kinase (PKC) was activated by arachidonic and myristic acids. This activation by both fatty acids required the calcium ion. Acidic phospholipid was also required for the activation by myristic acid, while that by arachidonic acid was inhibited by phospholipid.

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Upon stimulation of surface membranes of non-exitable cells, such as polymorphonuclear leukocytes and platelets, a number of metabolic changes are induced via a transmembrane control mechanism in which mobilization of Ca2+ in the biological membrane may play a critical role (1,3,9,11,14, 18, 24). Thus, the search for the triggering mechanism for intracellular metabolic activation has focussed on the determination of compounds by which the mobilization of cytoplasmic Ca2+ is controlled (6,8,10,16, 19, 22, 23). It is known that perturbation of surface membranes of these cells increases arachidonic acid metabolism and the turnover of phospholipids within the membrane/lipid bilayer, followed by release of their metabolites (4, 13, 15). Several investigators have suggested that certain oxidized metabolites (endoperoxides) of polyunsaturated long-chain fatty acids, such as arachidonic acid and its metabolites, increase membrane permeability for Ca2+ this effect is abolished by reducing the metabolites with SnCl2 (19, 20). Although the putative ionophoretic action of these metabolites for Ca2+ has received considerable attention, the biophysical functions of arachidonic acid and its metabolites have not been fully elucidated. Liposomes entrapped with Quin II, an indicator of Ca2+, have been used as a highly sensitive assay system for Ca2+ ionophoresis; formation of Quin II-Ca2+ complex markedly increases the fluorescence intensity at 490 nm (21). The present work demonstrates that several unsaturated long-chain fatty acids including arachidonic acid increase the translocation of Ca2+ across liposomal membrane.Ca2+ binds to Quin II in a stoichiometric ratio of one to one, and the fluorescence intensity is proportional to the logarithm of the concentration of Ca2+-Quin II complex. When CaCl2 (1 mM) was added to the incubation medium, the fluorescence Abbreviations used: DPPC, dipalmitoyl phosphatidyl choline; DCP, dicetyl phosphate.

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N‐hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM‐cellulose cation‐exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane‐associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+‐ and phospholipid‐dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.

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Although cholesterol is one of the major components of plasma membranes in eukaryotic cells, very little is known about its role in biological membranes. We reported previously (Okimasu et al., Cell Struct. Funct. 11, 273-283, 1986) that introduction of cholesterol into the liposomal membrane caused a decrease in membrane permeability, especially by the binding of cytoplasmic proteins to the liposomal membrane. The present study was carried out to further clarify the biochemical function of cholesterol in the membrane-protein interactions, especially under high osmotic pressure. The association of membranes with cytoplasmic proteins and their permeability were decreased by the introduction of cholesterol, but its effects were diminished in a hypertonic medium. The protein species associated with cholesterol-containing liposomes vary depending on the sort of hypertonic condition. It was suggested that since the degree of lipid packing by the cholesterol was reduced by the locally increased curvature in the lipid bilayer under high osmotic pressure, some cytoplasmic proteins can penetrate into the liposomal membrane.