Fatty acid acylation of rat brain myelin proteolipid protein in vitro: identification of the lipid donor.
The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.
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Bizzozero
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Lees
D019297:2,4-Dinitrophenol; D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D004140:Dinitrophenols / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism*; D005260:Female; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008297:Male; D009185:Myelin Proteins / Q000378:metabolism*; D018991:Myelin Proteolipid Protein; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D051381:Rats
D004140:Dinitrophenols; D005227:Fatty Acids; D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D010169:Palmitic Acids; D010171:Palmitoyl Coenzyme A; D019308:Palmitic Acid; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D008274:Magnesium; D019297:2,4-Dinitrophenol; D003065:Coenzyme A