L-3-hydroxyacyl coenzyme A dehydrogenase from pig heart muscle. EC 1.1.1.35 L-3-hydroxyacyl-CoA: NAD oxidoreductase.

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This chapter discusses the determination of L-3-hydroxyacyl coenzyme A dehydrogenase from pig heart muscle. This mitochondrial enzyme participates in the β-oxidation of fatty acids. It can be distinguished from acetoacetyl-CoA reductase (D-3-hydroxyacyl-CoA : NADP oxidoreductase) by the stereochemical configuration of the hydroxyacyl substrate and the principal cofactor utilized. The activity of the enzyme is routinely measured by monitoring the decrease in absorption of reduced nicotinamide adenine dicucleotide (NADH) at 340 nm, which accompanies the conversion of the S-acetoacetyl ester to the corresponding β-hydroxy compound. Either S-acetoacetylpantetheine or S-acetoacetyl-CoA can be used as substrate. However, because of expense and the more pronounced product inhibition encountered with the CoA ester, S-acetoacetylpantetheine is the substrate choice. The purification scheme described can be carried out with 80 hearts (20 kg). However, the initial stages are best performed in batches of 12–15 hearts. All buffers contained 1 m M EDTA and 2 m M β-mercaptoethanol, and the entire preparation was carried out at 0–4°. Specific activities are based on protein determined by the microbiuret reaction.

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关键词：

Animals Myocardium Hydroxy Acids Coenzyme A Alcohol Oxidoreductases Amino Acids Chromatography, Gel Chromatography, Ion Exchange Kinetics Methods