We showed that both PLP and PO are acylated in vivo and in vitro and that fatty acids are covalently linked to these proteins by an ester bond. However, it is not known if PLP and PO are acylated in the glial cell or in the myelin membrane. We have shown the presence of acid CoA ligase and acyltransferase in myelin isolated from the CNS which acylates PLP and DM-20 in a cell-free system. These studies are designed to ascertain if myelin isolated from the dysmyelinating quaking mice brain contains enzymes which will acylate PLP and DM-20 in a cell-free system. We will also study the cell-free acylation of PLP and DM-20 in myelin and myelin subfractions isolated from developing and mature rat brain to determine: a) if the developmental accumulation of PLP results in an increased incorporation of [3H]palmitic acid and [14C]palmitoyl CoA, b) if acylating enzymes are preferentially associated with myelin-like, light myelin and heavy myelin subfractions, c) if myristic acid is incorporated into these proteins at all stages of development and d) if myristic acid is attached to these proteins by ester or amide bond(s). Requirements for myelin PLP acyltransferase for optimal activity in a cell-free system will be determined. We will isolate peptide(s) from PLP acylated in vivo and in a cell-free system and determine the site(s) of attachment of fatty acids to specific amino acids. We will incubate myelin isolated from rat sciatic nerve with radiolabeled fatty acids with cofactors and radiolabeled CoA to determine if myelin contains the enzyme which will acylate PO in a cell-free system. These studies will be extended to examine the site(s) of acylation of PO in vitro and in a cell-free system. These studies will be extended to determine the specificity of acylation of soluble and membrane bound proteins after incubation of rat brain slices or sciatic nerve slices in vitro with palmitic acid and myristic acid. We will use cycloheximide to determine if inhibition of protein synthesis is related to the addition of fatty acids to the newly synthesized proteins.
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