Since discovery of a muscarinic receptor in myelin we have broadened this grant (reflected in title) to include the study of both receptors and enzymes which are intrinsic to the myelin membrane. Current work indicating that this receptor is linked to phosphoinositide metabolism will be continued to determine the details of phosphoinositide breakdown and resynthesis within myelin, as elicited by muscarinic agonists as well as depolarizing agents. A key enzyme in this process, phosphoinositide-specific phospholipase C, will be isolated from myelin and its properties studied. This will also be done with phosphatidylinositol synthetase, an enzyme presumed to be present in myelin because of its role in resynthesis of phosphoinositides. A third protein to be isolated from myelin is the GTP-binding protein which mediates the receptor-lined hydrolysis of phosphoinositides. Possible involvement of the axon in supplying subtrates for these reactions (as previously demonstrated for other myelin lipids) will be investigated by use of the optic system/axonal transport paradigm. Other substrates to be similarly studied are those required for sphingolipid synthesis, e.g. cerebrosides (enzymes involved in synthesis of the latter have been localized in myelin). These investigations will further establish the nature of the metabolic linkage between the axon and its myelin sheath. New myelin enzymes to be sought include those involved in sphingolipid synthesis, principally the lipophilic moieties. Since receptor- mediated breakdown of phosphoinositides leads to formation of diacylglycerol, an activator of protein kinase C, we shall attempt to identify myelin proteins (e.g. basic protein) whose phosphorylation is significantly affected by muscarinic agonists. Finally, we shall attempt to further characterize the adenylate cyclase-system which we recently discovered to be present in myelin.
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