Acylation of endogenous myelin proteolipid protein with different acyl-CoAs.
Fatty acyltransferase activity that catalyzes the transfer of palmitic acid from palmitoyl-CoA to the endogenous myelin proteolipid protein has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the acylation of proteolipid protein was obtained in 0.1% Triton X-100, 2 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.5 and at 37 degrees C. Other detergents had little or no effect on the reaction whereas acylation was completely abolished by sodium dodecyl sulphate (0.1%). Pulse-chase experiments indicated that the reaction involves the net addition of fatty acid to the protein and not a rapid fatty acid exchange. The rate of acylation was linear up to 30 min, indicating that the concentration of endogenous protein acceptor was constant. Under these conditions and at short time periods, the enzyme activity versus acyl-CoA concentration showed a hyperbolic curve. The apparent Km and Vmax for palmitoyl-CoA was 41 microM and 115 pmol/mg protein/min. Similar values were obtained for stearoyl and oleoyl-CoA, whereas myristoyl-CoA showed a lower specificity for the enzyme. The acyl-CoA specificity was also studied in competition experiments using several saturated and unsaturated fatty acid-CoAs. The product of the reaction was identified as myelin proteolipid protein and the fatty acid was shown to be attached to the protein via an ester linkage. Limited proteolysis and peptide mapping showed that the same sites on the proteolipid protein were acylated when the reaction was carried out in isolated myelin preparations or in brain tissue slices, suggesting physiological importance for the in vitro acylation of endogenous myelin proteolipid protein.
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000215:Acylation; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D044943:Fatty Acid Desaturases / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D009185:Myelin Proteins / Q000378:metabolism*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism*; D051381:Rats; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D000214:Acyl Coenzyme A; D003902:Detergents; D009185:Myelin Proteins; D011510:Proteolipids; D010171:Palmitoyl Coenzyme A; D044943:Fatty Acid Desaturases; D002918:Chymotrypsin; D014357:Trypsin