[Purification and characterization of Linoleoyl-CoA desaturase from rat liver microsomes (author's transl)].
Rat liver microsomes catalyzed the oxidative delta 6-desaturation of linoleoyl-CoA (C18: 2, delta 9.12.) to gamma-linolenoyl-CoA (c18: 3, delta 6.9.12.) by using molecular oxygen and NADH or NADPH as the electron donors. The antibodies against cytochrome b5 inhibited markedly the delta 6-desaturation in the intact microsomes of the rat liver, suggesting that cytochrome b5 participated in the delta 6-desaturation. These experimental results led us to the hypothesis that the delta 6-desaturation of linoleoyl-CoA followed the scheme. (See formula in text). Terminal "delta 6-desaturase" was purified from rat liver microsomes for the first time by Triton X-100 solubilization, DEAE-cellulose, CM-Sephadex and cytochrome b5-Sepharose chromatography using its high affinity for cytochrome b5. The final enzyme preparation was homogeneous when applied to sodium dodecyl sulfate disc gel electrophoresis. delta 6-desaturase appeared as a single polypeptide of 66,000 daltons containing 49% nonpolar amino acid residues and one atom of non-heme iron. We confirmed that delta 6-desaturase differed from delta 9-desaturase, which converted stearoyl-CoA to oleoyl-CoA. The delta 6-desaturase activity required NADH (or NADPH), linoleoyl-CoA, oxygen, lipid or detergent and three enzymes, such as NADH-cytochrome b5 reductase (or NADPH-cytochrome P -450 reductase), cytochrome b5, and delta 6-desaturase. The reconstituted system of these components also confirmed the electron flow represented in Scheme 1. The delta 6-desaturase activity was inhibited by iron chelators, cyanine and p-chloromercuriphenyl sulfonate. In the reconstituted system of Km value for linoleoyl-CoA was 47 micro M, the maximal velocity was 83nmol/min/mg protein of delta 6-desaturase and the optimal pH was 7.0. Catalase, superoxide dismutase and t-butanol showed supportive effects on the delta 6-desaturation of the reconstituted system when purified enzymes were employed.
D000214:Acyl Coenzyme A / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D002846:Chromatography, Affinity; D003580:Cytochromes / Q000276:immunology; D015786:Cytochromes b5; D003902:Detergents / Q000494:pharmacology; D004579:Electron Transport; D044943:Fatty Acid Desaturases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D066298:In Vitro Techniques; D050602:Linoleoyl-CoA Desaturase; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D051381:Rats; D012995:Solubility
D000214:Acyl Coenzyme A; D000596:Amino Acids; D000906:Antibodies; D003580:Cytochromes; D003902:Detergents; D009243:NAD; C026562:linoleoyl-coenzyme A; D009249:NADP; D015786:Cytochromes b5; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase