RESUMEN
This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed using the Baculovirus System localized in the microsomal fraction and catalyzed oxidation and reduction of the functional groups on steroids at carbons 3 and 17. Autoradiography assays revealed that the enzyme was most efficient as a 3alpha-hydroxysteroid oxidoreductase. High affinity of the enzyme for NADH (Km of 0.18 microM), lack of stereospecificity in the reductive direction, and poor efficiency for 3beta- versus 3alpha-hydroxyl oxidation could account for the observed transient accumulation of 3beta-stereoisomers in the oxidative reaction. Consistent with the 65% sequence identity with RoDH dehydrogenases, the enzyme oxidized all-trans-retinol with the Km value of 3.2 microM and Vmax value of 1.2 nmol/min per milligram microsomes. 13-cis-Retinol and all-trans-retinol bound to the cellular retinol-binding protein were not substrates. Neurosteroid allopregnanolone was a better substrate than all-trans-retinol with the Km and Vmax values of 0.24 microM and 14.7 nmol/min per milligram microsomes. Northern blot analysis revealed that the corresponding mRNA was present in adult human brain (caudate nucleus, amygdala, hippocampus, substantia nigra, thalamus) and spinal cord in addition to other tissues. The message was also detected in fetal lung, liver, and brain. Antibodies against the enzyme recognized a protein of approximately 35 kDa in the particulate fraction of human tissues. This study presents new information about enzymatic properties, substrate specificity, and tissue distribution of this enzyme, and provides a better insight into its possible physiological function(s).
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/química , Microsomas/enzimología , 3-Hidroxiesteroide Deshidrogenasas/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Baculoviridae/metabolismo , Northern Blotting , Western Blotting , Encéfalo/embriología , Encéfalo/metabolismo , Carbono/química , Línea Celular , Clonación Molecular , Humanos , Insectos , Cinética , Hígado/embriología , Pulmón/embriología , Masculino , Modelos Químicos , Oxígeno/metabolismo , Próstata/enzimología , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Testículo/enzimología , Factores de Tiempo , Distribución Tisular , Vitamina A/farmacologíaRESUMEN
We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons. The recombinant protein produced using the baculovirus system is localized in the microsomal fraction of Sf9 cells and is an integral membrane protein with cytosolic orientation of its catalytic domain. The enzyme exhibits an oxidoreductase activity toward hydroxysteroids with NAD(+) and NADH as the preferred cofactors. The enzyme is most efficient as a 3alpha-hydroxysteroid dehydrogenase, converting 3alpha-tetrahydroprogesterone (allopregnanolone) to dihydroprogesterone and 3alpha-androstanediol to dihydrotestosterone with similar catalytic efficiency (V(max) values of 13-14 nmol/min/mg microsomal protein and K(m) values of 5-7 microm). Despite approximately 44-47% sequence identity with retinol/3alpha-hydroxysterol dehydrogenases, the enzyme is not active toward retinols. The corresponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain, heart, colon, testis, placenta, lung, and lymph node. Thus, the new short chain dehydrogenase represents a novel type of microsomal NAD(+)-dependent 3alpha-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Microsomas/enzimología , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , ADN Complementario , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The present paper is a review of up-to-date findings on vitamin A transport from intestines to target-cell and metabolism of this fat-soluble vitamin. The hypotheses of possible enzymes participation in the process of etherification/hydrolysis and oxidation/reduction of vitamin A are discussed. Furthermore, possible roles of cellular retinoid-binding proteins in the process of vitamin A transport and metabolism are reviewed.
Asunto(s)
Vitamina A/metabolismo , Transporte Biológico , Sistema Digestivo/metabolismo , Humanos , Absorción Intestinal , Hígado/citología , Hígado/metabolismo , Vitamina A/químicaRESUMEN
It has been established that under intake of small doses of 137Cs by rat for 9 months the radioactivity of whole rat body was increased in irregular manner. Under this condition the level of fat-soluble vitamins A and E is decreased and the decrease is well correlated with a level of radionuclide accumulation by rat's body. The possible causes of decrease of the vitamins A and E level under intake of small doses of radionuclide are discussed.
Asunto(s)
Ácido Ascórbico/análisis , Hígado/efectos de la radiación , Vitamina A/análisis , Animales , Radioisótopos de Cesio , Relación Dosis-Respuesta en la Radiación , Hígado/química , Masculino , RatasRESUMEN
The retinol interaction with intact cellular nuclei, nuclear envelope and chromatin was investigated. We have shown that the Cellular Retinol-Binding Protein (CRBP) plays a very important role in such interaction. Retinol can specifically interacts with nuclei, nuclear envelope and chromatin only when it presents as a complex with CRBP. The obtained data allowed us to suggest that the process of retinol delivery to hypothetical nuclear receptors include of at least the following stages: 1) specific binding with nuclear envelope; 2) penetration into nuclei; 3) specific binding with chromatin receptors. CRBP is a necessary component at all this stages. Also we show that CRBP has not a species or tissue specificity.
Asunto(s)
Núcleo Celular/metabolismo , Vitamina A/metabolismo , Transporte Biológico , Cromatina/metabolismo , Membrana Nuclear/metabolismo , Unión Proteica , Proteínas de Unión al Retinol/metabolismo , Proteínas Celulares de Unión al RetinolRESUMEN
Interaction of the 3H retinol-cellular retinol-binding protein complex with nuclei is shown. The uptake process is specific and saturable. Free retinol does not penetrate to a nucleus. An assumption is advanced that cellular retinol-binding protein with molecular weight 14 kDa has no specific tissues and is involved in the transport of retinol to the nucleus and to the Golgi apparatus of the glandular stomach mucosa cells.
Asunto(s)
Núcleo Celular/metabolismo , Mucosa Gástrica/metabolismo , Proteínas de Unión al Retinol/química , Vitamina A/química , Animales , Pollos , Mucosa Gástrica/ultraestructura , Masculino , Unión Proteica , Proteínas de Unión al Retinol/metabolismo , Proteínas Celulares de Unión al Retinol , Vitamina A/metabolismoRESUMEN
Cellular retinol-binding protein from bovine liver has been purified to homogeneity. The protein binds retinol with high affinity; the apparent dissociation constant was determined by fluorometric titration to be 2.18 x 10(-3) M. Retinol bound to the protein has an absorption spectrum (lambda max = 350 nm) and considerably differs from the spectrum of retinol absorption in ethanol (lambda max = 325 nm). The protein is a single polypeptide chain with a molecular weight of approximately 14 kDa based on information obtained by sodium dodecyl sulfate-polyacrylamide electrophoresis.