CHARACTERIZATION OF THE MICROSOMAL AND PARTIALLY PURIFIED RETINAL REDUCTASE OF RAT SMALL-INTESTINE

A retinal reductase activity Sound in rat small intestinal mucosa Mns characterized as a partially purified preparation and as present in a preparation of microsomes, Stereochemical studies revealed that hydrog en,vas added to the re face of the aldehyde group during reduction of retinal by the microsomal activity, matching the stereochemistry of re duction by isolated intestinal segments. Retinal reduction by this enz yme was determined to a sequential reaction, most likely with reduced nicotinamide adenine dinucleotide (NADH) binding first. The enzyme was specific for retinal and did not oxide retinol to a measurable extent in the presence of cellular retinol-binding protein, type II (CRBP II ). The presence of greater than stoichiometric amounts of CRBP II did not substantially alter the rate of retinal reduction, however. The Mi chaelis constants for both the bound and unbound forms of the substrat e, rather than the small amount of free retinal in equilibrium with th e binding protein, indicating direct interaction between the enzyme an d CRBP II-retinal. Competition experiments indicated increased recogni tion of the binding protein by the enzyme when the protein was in the ''holo'' form, with a preference for CRBP II over cellular retinol-bin ding protein (CRBP), a very similar binding protein found in other cel ls types. The properties determined strongly suggest this is the enzym e involved in the processing of beta-carotene-derived retinal after up take by the absorptive cell.