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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS013464-12
Application #
3395192
Study Section
Neurology B Subcommittee 1 (NEUB)
Project Start
1976-12-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1991-03-31
Support Year
12
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Agrawal, H C; Agrawal, D (1991) Proteolipid protein and DM-20 are synthesized by Schwann cells, present in myelin membrane, but they are not fatty acylated. Neurochem Res 16:855-8
Agrawal, H C; Agrawal, D; Strauss, A W (1990) Cleavage of the P0 glycoprotein of the rat peripheral nerve myelin: tentative identification of cleavage site and evidence for the precursor-product relationship. Neurochem Res 15:993-1001
Agrawal, H C; Sprinkle, T J; Agrawal, D (1990) 2',3'cyclic nucleotide-3'-phosphodiesterase in peripheral nerve myelin is phosphorylated by a phorbol ester-sensitive protein kinase. Biochem Biophys Res Commun 170:817-23
Agrawal, H C; Noronha, A B; Agrawal, D et al. (1990) The myelin-associated glycoprotein is phosphorylated in the peripheral nervous system. Biochem Biophys Res Commun 169:953-8
Agrawal, H C; Sprinkle, T J; Agrawal, D (1990) 2',3'-cyclic nucleotide-3'-phosphodiesterase in the central nervous system is fatty-acylated by thioester linkage. J Biol Chem 265:11849-53
Agrawal, H C; Agrawal, D (1989) Effect of cycloheximide on palmitylation of PO protein of the peripheral nervous system myelin. Biochem J 263:173-7
Agrawal, H C; Agrawal, D (1989) Tumor promoters accentuate phosphorylation of PO: evidence for the presence of protein kinase C in purified PNS myelin. Neurochem Res 14:409-13
Bourdette, D N; Seil, F J; Meshul, C K et al. (1988) Antisera to an axolemma-enriched fraction have antiaxon and antimyelin effects in vitro. Ann N Y Acad Sci 540:423-6
Yoshimura, T; Agrawal, D; Agrawal, H C (1987) Cell-free acylation of rat brain myelin proteolipid protein and DM-20. Biochem J 246:611-7
Agrawal, H C; Agrawal, D; Yoshimura, T et al. (1987) In vitro acylation of myelin PLP and DM-20 in the quaking mouse brain. Neurochem Res 12:783-6

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