Purification and characterization of alpha,beta-ketoalkene double bond reductases from bovine eyes.
OBJECTIVE
To explore the alpha,beta-ketoalkene double bond reductases responsible for xenobiotic metabolism in bovine ocular tissues using trans-phenyl-l-propenyl ketone as a model substrate.


METHODS
A mixture of trans-phenyl-l-propenyl ketone, NADPH or NADH, and an enzyme source in 0.1 M K,Na-phosphate buffer (pH 7.4) was incubated for 15 min at 37 degrees C. Phenyl propyl ketone formed was quantified by high performance liquid chromatography (HPLC).


RESULTS
The lens, ciliary body, iris, retinal pigment epithelium (RPE)-choroid, retina, and cornea exhibited alpha,beta-ketoalkene double bond reductase activities in the presence of NADPH or NADH. An alpha,beta-ketoalkene double bond reductase was purified to homogeneity from the iris-ciliary body cytosol. The molecular weight was estimated to be 58,000 by gel filtration, and 40,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was inhibited by dicumarol, quercitrin, indomethacin, disulfiram, and p-chloromercuribenzoic acid. The enzyme exhibited double bond reductase activity toward 2-alkenals as well as alpha,beta-ketoalkenes. Another alpha,beta-ketoalkene double bond reductase was also purified to homogeneity from the lens cytosol. The molecular weight was estimated to be 105,000 by gel filtration and 40,000 by SDS-PAGE. The enzyme activity was inhibited by dicumarol and quercitrin. The enzyme exhibited double bond reductase activity toward some alpha,beta-ketoalkenes.


CONCLUSIONS
Two kinds of enzymes responsible for reduction of the carbon-carbon double bond of xenobiotics were purified for the first time from ocular tissues. The molecular weights, substrate specificities, and sensitivities to inhibitors of the enzymes were different from each other.
Takeda
Y
Y
Department of Ophthalmology, Hiroshima University School of Medicine, Japan.
Mishima
H K
HK
Kitamura
S
S
Tatsumi
K
K
D000475:Alkenes / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002924:Ciliary Body / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005123:Eye / Q000201:enzymology*; D007498:Iris / Q000201:enzymology; D007908:Lens, Crystalline / Q000201:enzymology; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification*; D013268:Stimulation, Chemical; D013379:Substrate Specificity; D014018:Tissue Distribution
D000475:Alkenes; D009243:NAD; D009249:NADP; D010088:Oxidoreductases