Spectrophotometric assay of 2,4-dienoyl coenzyme A reductase with 5-phenyl-2,4-pentadienoyl-coenzyme A as substrate.
The spectrophotometric assay of 2,4-dienoyl coenzyme A (CoA) reductase (EC 1.1.1.34) was modified to improve the linearity and sensitivity of this method. 5-Phenyl-2,4-pentadienoyl-CoA, which has an absorbance maximum at 340 nm with an extinction coefficient of 44,300 M-1 cm-1, was synthesized and used as substrate. This compound is reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent 2,4-dienoyl-CoA reductase to 5-phenyl-3-pentenoyl-CoA. When a tissue homogenate serves as an enzyme source, the product is further metabolized by delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) to 5-phenyl-2-pentenoyl-CoA, which is hydrated to 5-phenyl-3-hydroxypentanoyl-CoA by enoyl-CoA hydratase (EC 4.2.1.17). The modified assay method, which measures the decrease in absorbance at 340 nm due to the reduction of 5-phenyl-2,4-pentadienoyl-CoA and the oxidation of NADPH, is linear for a longer period of time and is twice as sensitive as the conventional assay with 2,4-decadienoyl-CoA as substrate.
Department of Chemistry, City College of the City University of New York, New York 10031.
M A
MA
Nada
K
K
Shoukry
H
H
Schulz
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D044943:Fatty Acid Desaturases / Q000032:analysis* / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats; D013053:Spectrophotometry; D013379:Substrate Specificity
C090296:5-phenyl-2,4-pentadienoyl-coenzyme A; D000214:Acyl Coenzyme A; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase