Specific assay of medium-chain acyl-CoA dehydrogenase based on the spectrophotometric measurement of product formation.
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-phenylpropionyl-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 acyl-CoA 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 cinnamoyl-CoA 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.
Yao
K W
KW
Department of Chemistry, City College of the City University of New York, New York 10031.
Schulz
H
H
D000214:Acyl Coenzyme A / Q000032:analysis; D042964:Acyl-CoA Dehydrogenase; D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000032:analysis*; D000818:Animals; D007962:Leukocytes / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D009206:Myocardium / Q000201:enzymology; D051381:Rats; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D000214:Acyl Coenzyme A; C022903:cinnamoyl-coenzyme A; D042964:Acyl-CoA Dehydrogenase; D044942:Acyl-CoA Dehydrogenase, Long-Chain