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Preparation and Properties of Retinal-oxidizing Enzyme from Rat Intestinal Mucosa

From the ¹ From the West Virginia University School of Medicine Department of Biochemistry, Morgantown, West Virginia 26506

An enzyme which converts retinal to retinoic acid was purified from rat intestinal mucosa approximately 160-fold via a combination of acetone precipitation, ammonium sulfate precipitation, and DEAE-cellulose ion exchange chromatography. The purified preparation appeared to be homogeneous in the ultracentrifuge, on ion exchange chromatography, and on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approximately 80,000 and its isoelectric point was in the neighborhood of 6.7. The enzyme preparation contained approximately 2 moles of iron per mole of enzyme. The absorption spectrum of the enzyme indicated a maximum at 280 mµ and a much smaller band around 410 mµ.

Purified retinal-oxidizing enzyme from rat intestinal mucosa stoichiometrically and irreversibly converted retinal to retinoic acid. The latter was identified as the product of the reaction by ultraviolet absorption spectrum, thin layer chromatography, and gas chromatography. The product was approximately 96% pure as determined by its E^1%_{1 cm} value.

The enzymatic reaction exhibited Michaelis-Menten kinetics with a K_m of approximately 3.0 x 10^-4 m. The rate of the reaction was constant for 120 min, directly proportional to the enzyme concentration, and maximal when GSH, NAD, FAD, and Fe²⁺ were added. The pH optimum was approximately 7.7. The reaction proceeded well under both aerobic and anaerobic conditions. The enzyme acted upon both the all-trans and 13-cis forms of retinal.

Reduced nicotinamide adenine dinucleotide noncompetitively inhibited the reaction and was found to exhibit a control over the utilization of retinal in vitro. Thiols and metal ions stimulated the reaction, while thiol inhibitors and chelators inhibited the reaction. It is suggested that retinal-oxidizing enzyme is a metalloprotein which requires sulfhydryl groups for maximal enzymic activity.

In the presence of H₂¹⁸O, the oxidation of retinal by retinaloxidizing enzyme appeared to resemble a dehydrogenase rather than an oxygenase or oxidase.

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