Properties of Short-Chain Dehydrogenase/Reductase RalR1:  Characterization of Purified Enzyme, Its Orientation in the Microsomal Membrane, and Distribution in Human Tissues and Cell Lines

Abstract

Recently, we reported the first biochemical characterization of a novel member of the short-chain dehydrogenase/reductase superfamily, retinal reductase 1 (RalR1) (Kedishvili et al. (2002) J. Biol. Chem. 277, 28909-28915). In the present study, we purified the recombinant enzyme from the microsomal membranes of insect Sf9 cells, determined its catalytic efficiency for the reduction of retinal and the oxidation of retinol, established its transmembrane topology, and examined the distribution of RalR1 in human tissues and cell lines. Purified RalR1-His(6) exhibited the apparent K(m) values for all-trans-retinal and all-trans-retinol of 0.12 and 0.6 microM, respectively. The catalytic efficiency (k(cat)/K(m)) for the reduction of all-trans-retinal (150,000 min(-1) mM(-1)) was 8-fold higher than that for the oxidation of all-trans-retinol (18,000 min(-1) mM(-1)). Protease protection assays and site-directed mutagenesis suggested that the enzyme is anchored in the membrane by the N-terminal signal-anchor domain, with the majority of the polypeptide chain located on the cytosolic side of the membrane. An important feature that prevented the translocation of RalR1 across the membrane was the positively charged R(25)K motif flanking the N-terminal signal-anchor. The cytosolic orientation of RalR1 suggested that, in intact cells, the enzyme would function predominantly as a reductase. Western blot analysis revealed that RalR1 is expressed in a wide variety of normal human tissues and cancer cell lines. The expression pattern and the high catalytic efficiency of RalR1 are consistent with the hypothesis that RalR1 contributes to the reduction of retinal in various human tissues.

Kinetic Analysis of Human Enzyme RDH10 Defines the Characteristics of a Physiologically Relevant Retinol Dehydrogenase

“…This PCR product was subcloned between the XbaI and HindIII sites of pET28a vector (Novagen, Madison, WI) as an intermediate step before transferring the RDH10 cDNA into the SmaI-NotI sites of the pVL1393 vector modified as described previously (18,19). The final construct of RDH10 in pVL1393-encoded RDH10 fused with the His 6 tag on the C terminus.…”

“…Determination of kinetic constants of RDH10 was performed as described previously in detail for RDH12 and RDH11 (18,19). All-trans-retinol, all-trans-retinaldehyde, and 9-cis-retinaldehyde were obtained from Sigma and purified by highpressure liquid chromatography (HPLC).…”

“…The amount of protein varied between 3 and 20 g, so that the amount of the product did not exceed 10% of the substrate. Reaction rates were determined based on the percent substrate conversion as described previously (18,19). Kinetic constants were calculated using GraFit (Erithacus Software Ltd., UK) and expressed as the mean Ϯ S.D.…”

“…Retinoids were extracted into hexane (24) and separated by normal phase HPLC using the Spherisorb S3W column as described above except that the linear mobile phase consisted of 99.6:0.3:0.1 hexane:isopropyl alcohol:acetic acid. Peaks were identified by comparison to retention times of retinoid standards and evaluation of wavelength maxima and quantified as described previously (18,19).…”

“…Western Blot Analysis-The protocol for Western blot analysis was described elsewhere (18). RDH10-His 6 expression in Sf9 cells was detected using His tag monoclonal mouse antibody (Novagen, Madison, WI) at a 1:6,000 dilution.…”