No abstract

All-trans-retinoic acid is a metabolite of vitaminAll-trans-retinoic acid is a metabolite of vitamin A (all-transretinol) that functions as an activating ligand for a family of nuclear retinoic acid receptors (1). In target tissues, all-transretinoic acid is produced by the oxidation of all-trans-retinaldehyde catalyzed by cytosolic aldehyde dehydrogenases (Fig.

All-trans-retinol is the precursor for all-trans-retinoic acid, the activating ligand for nuclear transcription factors retinoic acid receptors. In the cytosol of various cells, most retinol exists in a bound form, complexed with cellular retinol binding protein type I (holo-CRBP). Whether retinoic acid is produced from the free or bound form of retinol is not yet clear. Here, we present evidence that holo-CRBP is recognized as substrate by human microsomal short-chain dehydrogenase/reductase (SDR) RoDH-4 with the K(m) value close to the liver concentration of holo-CRBP. The ability to utilize holo-CRBP differentiates RoDH-4 from a related enzyme, RoDH-like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), which is 3-fold more active with free retinol than RoDH-4 but is 15-fold less active toward holo-CRBP. Recognition of the cytosolic holo-CRBP as substrate is consistent with RoDH-4 orientation in the membrane. As established by immunoprecipitation and glycosylation scanning, RoDH-4 faces the cytosolic side of the membrane. Purified RoDH-4, stabilized by reconstitution into proteoliposomes, exhibits the apparent K(m) values for substrates and NAD(+) similar to those of the microsomal enzyme and oxidizes holo-CRBP with the catalytic efficiency (k(cat)/K(m)) of 59 min(-1) mM(-1). Apo-CRBP acts as a strong competitive inhibitor of holo-CRBP oxidation with an apparent K(i) value of 0.2 microM. The results of this study suggest that the human retinol-active SDRs are not functionally equivalent and that, in contrast to RoDH-like 3alpha-HSD, RoDH-4 can access the bound form of retinol for retinoic acid production and is regulated by the apo-/holo-CRBP ratio.

No abstract

The aim of our investigation was to study the red blood cell (RBC) membrane effects of NaNO(2)-induced oxidative stress. Hyperpolarization of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. These membrane changes preceded reduced glutathione depletion and were observed simultaneously with methemoglobin (metHb) formation. Changes of the glutathione pool (total and reduced glutathione, and mixed protein-glutathione disulfides) during nitrite-induced erythrocyte oxidation have been demonstrated. The rates of intracellular oxyhemoglobin and GSH oxidation highly increased as pH decreased in the range of 7.5-6.5. The activation energy of intracellular metHb formation obtained from the temperature dependence of the rate of HbO(2) oxidation in RBC was equal to 16.7+/-1.6 kJ/mol in comparison with 12.8+/-1.5 kJ/mol calculated for metHb formation in hemolysates. It was found that anion exchange protein (band 3 protein) of the erythrocyte membrane does not participate significantly in the transport of nitrite ions into the erythrocytes as band 3 inhibitors (DIDS, SITS) did not decrease the intracellular HbO(2) oxidation by extracellular nitrite.