Purification and Properties of Rat Liver Microsomal Stearyl Coenzyme A Desaturase

Abstract

AB STRACTThe terminal enzyme of the NADHdependent stearyl coenzyme A desaturase system has been isolated from rat liver microsomes. This desaturase is a single polypeptide of 53,000 daltons containing 62% nonpolar amino-acid residues and one atom of non-heme iron. The purified protein forms high molecular weight aggregates that can be dispersed by detergent procedures.Desaturase activity requires NADH, stearyl coenzyme A, oxygen, lipid, and the three enzymes, cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and desaturase. Cytoehrome b5 is the direct electron donor to the desaturase, which appears to utilize the iron in the oxidationreduction sequence during desaturation of stearyl coenzyme A.The characterization of the liver microsomal stearyl coenzyme A desaturase system by workers in several laboratories (1-11), using spectral methods and partially purified components from hen and rat liver microsomes has shown that the oxygen-and NADH-dependent formation of oleyl coenzyme A involves cytochrome b5 reductase (EC 1.6.2.2; NADH : ferricytochrome b5 oxidoreductase), cytochrome b5, and a terminal, cyanidesensitive oxidase, the desaturase. The present report describes the isolation of the terminal component, the desaturase, from liver of rats induced for this enzyme. A sequence of extractions of contaminating proteins from microsomes, followed by solubilization of the desaturase by detergents and subsequent purification, results in a protein preparation that appears to be homogeneous by several criteria. It contains one molecule of non-heme iron, which appears to participate in stearyl coenzyme A desaturation in the presence of oxygen and reduced cytochrome b5. METHODS AND MATERIALSSodium deoxycholate (Matheson, Coleman and Bell) was purified as described (12). Sephadex G-75 was obtained from Pharmacia, and Whatman DEAE-cellulose (DE-52), from Reeve Angel. Triton X-100 was the purified product of Packard Instrument Co. The ['4C]deoxycholate was obtained from Mallinckrodt Chemical Works, the NADH, stearyl coenzyme A, and bathophenanthroline sulfonate were products of Sigma Chemical Co., and [14C]stearyl coenzyme A was a product of New England Nuclear. The egg lecithin was obtained from Supelco in a chloroform solution. We prepared clear liposomes by removing solvent in a nitrogen stream, adding 0.02 M Tris-acetate buffer, pH 8.1 (Tris buffer), to yield a suspension of 12.5 mg/ml, sonicating under nitrogen at 00 for 15-20 min at 40 mW, and centrifuging the slightly turbid suspension for 15 min at 100,000 X g to yield the clear liposome suspension, which was stored under nitrogen at 00 and used for no longer than 10 days. Abbreviation: Tris buffer, Tris-acetate buffer (pH 8.1). 4565Cytochrome b5 was prepared from either calf or rabbit liver microsomes by the detergent extraction procedure that yields the complete form of this heme protein (13). The proteins from the two species were indistinguishable as electron donors for the desaturase. The soluble catalytic fragment of cytochrome b5 was prepared by tryptic dige...

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