Abstract
A spectrophotometric method for assaying medium-chain acyl-CoA dehydrogenase is described. The assay measures at 308 nm the formation of cinnamoyl-CoA from 3-phenylpropionyl-CoA in the presence of phenazine methosulfate as electron acceptor. Apparent kinetic constants (Km, Vmax) determined with 3-phenyl-propionyl-CoA are similar to constants obtained with octanoyl-CoA, the preferred substrate of this enzyme. The assay is specific for medium-chain acyl-CoA dehydrogenase because long-chain and short-chain acylCoA dehydrogenases exhibit little or no activity with 3-phenylpropionyl-CoA as substrate. Since absorbance changes at 308 nm caused by other reactions are less than 5% of the absorbance change due to cinnamoylCoA formation catalyzed by medium-chain acyl-CoA dehydrogenase, the assay can be used to measure the activity of this enzyme in crude tissue homogenates. Specific activities of medium-chain acyl-CoA dehydrogenase determined by use of this assay in homogenates of rat liver, heart, and leukocytes were found to be 29, 68, and 2.1 mU/mg of protein, respectively.
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