The Enzymatic Reduction of Retinal to Retinol in Rat Intestine

The reduction of retinal to retinol was examined with enzyme preparations from homogenates of rat intestinal mucosa. Retinal reduction was catalyzed by a soluble mucosal enzyme which was purified 13-fold by ammonium sulfate precipitation, column chromatography, and heating (55° for 6 min). The enzyme was relatively heat-stable and had a molecular weight approximately in the range of 60,000 to 80,000. The partly purified reductase was unable to oxidize ethanol in the presence of NAD+. Retinal reduction required NADH or NADPH as cofactor. Both reduced nucleotides were effective; at low nucleotide concentration NADH was more effective, whereas at high concentrations the reaction rate was slightly greater with NADPH. The reaction was stimulated by the addition of glutathione and was inhibited by —SH inhibitors. No other cofactors were required. There was a sharp pH optimum near 6.3. Retinal reduction displayed typical Michaelis kinetics, with a Vmax of 2 x 10-6 moles of retinal formed per hour per mg of protein and with an apparent Km of 2 x 10-5 m. The enzyme appears to be a relatively nonspecific aldehyde reductase. Short and medium chain aliphatic aldehydes, of length C-2 to C-14, were actively reduced, with greatest activity being seen with aldehydes of length C-4 to C-8. Unsaturated C-18 fatty aldehydes were reduced at a lesser rate, but saturated aldehydes of length C-16 or greater were not reduced. The enzyme was stereospecific for 4R-NADH-4-3H1, and did not incorporate tritium from 4S-NADH-4-3H1 into the product retinol.