Prior studies indicate that bacterial lipopolysaccharides (LPS) prime macrophages for enhanced secretion of arachidonic acid (20:4) metabolites when subsequently challenged with a spectrum of triggers. LPS promotes the covalent attachment of myristic acid to specific macrophage proteins. The time and concentration dependence of LPS-induced protein myristoylation is consistent with a role for myristoylation in LPS-priming of the 20:4 cascade. The central hypothesis which we shall test proposes that LPS primes macrophages for enhanced 20:4 secretion by promoting the myristoylation of key proteins involved in the regulation of 20:4 metabolism. The myristoylation of these proteins promotes their association with membrane bound substrates and consequently, ensures more efficient catalysis during the mobilization and oxygenation of 20:4. LPS analogs with defined structures will be used to determine structural requirements for LPS-dependent priming of 20:4 metabolism, triggering of the 20:4 cascade, protein myristoylation and protein kinase C activation. The influence of LPS on lipocortin activity will be determined. Cell- free reconstitution of 20:4 metabolism will be attempted and the influence of LPS on the activity of the enzymes of the 20:4 cascade will be examined. We will examine whether there is a correlation in the subcellular distribution of the enzymes of the 20:4 cascade and the myristoylated proteins. We will attempt to identify the unknown stimulus-induced myristoylated proteins by immunoprecipitation with specific antisera. One of the proteins whose myristoylation is induced by LPS is a major specific substrate of protein kinase C. We will characterize the myristoylation and phosphorylation of the 68 kDa specific protein kinase C substrate and examine whether these covalent modifications influence its subcellular location. The fate of myristic acid within the cellular lipid pool will be investigated. We will determine whether LPS derived fatty acids are covalently transferred to proteins. Synthetic peptide substrates will be used to establish a cell free assay for myristoyltransferase(s) activity which will facilitate the identification of this important regulatory enzyme(s). We have also shown that the macrophage activating factor interferon-gamma induces the myristoylation of a single polypeptide of Mr 48 kDa within 2 hr. This represents one of the earliest known responses to IFN gamma and may have a role in the activation of macrophages for enhanced microbiocidal and tumoricidal capacity. The IFN gamma induced myristoylation reaction will be characterized.
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