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

Academic Article

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-His6 exhibited the apparent K m values for all-trans-retinal and all-trans-retinol of 0.12 and 0.6 μM, respectively. The catalytic efficiency (kcat/Km) 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 R25K 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.
  • Authors

    Published In

  • Biochemistry  Journal
  • Digital Object Identifier (doi)

    Author List

  • Belyaeva OV; Stetsenko AV; Nelson P; Kedishvili NY
  • Start Page

  • 14838
  • End Page

  • 14845
  • Volume

  • 42
  • Issue

  • 50