Solubilization and purification of hepatic microsomal trans-2-enoyl-CoA reductase: evidence for the existence of a second long-chain enoyl-CoA reductase.
The present study describes the solubilization and purification of a NADPH-specific trans-2-enoyl-CoA reductase from rat liver microsomes. The final preparation was purified to near homogeneity and had a minimal molecular weight of 51,000 +/- 2,000, as judged by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. This enzyme specifically used NADPH, as cofactor, and was chromatographically (2',5'-ADP-agarose) separated from another trans-2-enoyl-CoA reductase which utilized either NADH or NADPH as cofactor. The NADPH-specific trans-2-enoyl-CoA reductase catalyzed the reduction of trans-2-enoyl-CoAs from 4 to 16 carbon units. The Km values for crotonyl-CoA, trans-2-hexenoyl-CoA, and trans-2-hexadecenoyl-CoA were 20, 0.5, and 1.0 microM, while the Km value for NADPH was 10 microM. Although N-ethylmaleimide, heat treatment, and limited proteolysis with trypsin affected the reduction of short-chain (C4) and long-chain (C16) substrates equally, and in spite of the fact that a single protein band was observed on SDS-gels, at the present time one cannot state unequivocally that the purified preparation contained only one reductase. trans-2-Hexenoyl-CoA, for example, did not inhibit the reduction of trans-2-hexadecenoyl-CoA to palmitoyl-CoA and trans-2-decenoyl-CoA to decanoyl-CoA whereas it strongly inhibited the conversion of crotonyl-CoA to butyryl-CoA. The potential implications of this finding are discussed. Finally, the reductase preparation was shown not to contain either heme, nonheme iron, or a flavin prosthetic group.
D000214:Acyl Coenzyme A / Q000378:metabolism; D044944:Acyl-CoA Dehydrogenases; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D044943:Fatty Acid Desaturases / Q000145:classification / Q000302:isolation & purification*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D009249:NADP; C010701:crotonyl-coenzyme A; D044943:Fatty Acid Desaturases; D044944:Acyl-CoA Dehydrogenases; C100154:acyl-CoA dehydrogenase (NADP+)