PURIFICATION AND CHARACTERIZATION OF ALPHA,BETA-KETOALKENE DOUBLE-BOND REDUCTASES FROM BOVINE EYES

Purpose. To explore the alpha,beta-ketoalkene double bond reductases r esponsible for xenobiotic metabolism in bovine ocular tissues using tr ans-phenyl-1-propenyl ketone as a model substrate. Methods. A mixture of trans-phenyl-1-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 3 7 degrees C. Phenyl propyl ketone formed was quantified by high perfor mance liquid chromatography (HPLC). Results. The lens, ciliary body, i ris, retinal pigment epithelium (RPE)-choroid, retina, and cornea exhi bited alpha,beta-ketoalkene double bond reductase activities in the pr esence of NADPH or NADH. An alpha,beta-ketoalkene double bond reductas e 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, ind omethacin, disulfiram, and p-chloromercuribenzoic acid. The enzyme exh ibited double bond reductase activity toward 2-alkenals as well as alp ha,beta-ketoalkenes. Another alpha,beta-ketoalkene double bond reducta se was also purified to homogeneity from the lens cytosol. The molecul ar weight was estimated to be 105,000 by gel filtration and 40,000 by SDS-PAGE. The enzyme activity was inhibited by dicumarol and quercitri n. The enzyme exhibited double bond reductase activity toward some alp ha,beta-ketoalkenes. Conclusions. Two kinds of enzymes responsible for reduction of the carbon-carbon double bond of xenobiotics were purifi ed for the first time from ocular tissues. The molecular weights, subs trate specificities, and sensitivities to inhibitors of the enzymes we re different from each other.