ROLES OF PHOSPHOLIPID AND CA2+ IN BRAIN

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The overall goals of this renewal application cover primarily the molecular, immunological, pharmacological and regulatory aspects of the recently-identified phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) system in brain. The specific aims are as follows: (1) PL-Ca-PK, the enzyme. Attempts will be made to improve the purification of the enzyme from pig or bovine brain. Similarly, production of monoclonal antibodies to the enzyme will be improved, and the distribution of the enzyme in brain will be studied immunocytochemically. (2) Endogenous substrates. The sites of phosphorylation in myelin basic protein (MBP) by PL-Ca-PK will be determined. Once this is done, the minimal structural determinants of the substrate specificity for PL-Ca-PK will be explored using synthetic oligopeptides. The effects of phosphorylation of MBP and its fragments or peptides on their ability or inability to induce experimental allergic encephalomyelitis will be explored. In addition to MBP, a number of other endogenous substrates for PL-Ca-PK in brain will be further investigated. (3) Regulation of PL-Ca-PK system. Regulations by various agents of the enzyme activity, the phosphorylation state of endogenous substrates and possible "translocation" of PL-Ca-PK in brain will be investigated. (4) A new S-100-modulated protein phosphorylation system. The S-100-modulated enzyme (protein kinase X) and its l9K Mr substrate protein in rat brain will be further purified and characterized. It is hoped that the proposal will produce new knowledge regarding regulation of brain biology and pathology by phospholipid, Ca2+ and S-100 protein via protein phosphorylation reaction.