Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The conversion of testosterone to 5 alpha-dihydrotestosterone, catalysed by 4-ene-steroid 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase EC 1.3.1.22) requires NADPH. In the present study, the role of flavins and Co-enzyme Q in this proton transfer was investigated for the first time in any male androgen target tissue. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) inhibited epididymal nuclear 4-ene-steroid 5 alpha-reductase activity non-competitively with respect to the substrate testosterone. However, neither the oxidized nor reduced forms of Co-enzyme Q affected the Kmapp or the Vmaxapp and the reduced form was unable to support catalytic activity in the absence of NADPH. Further investigation of the effects of flavins revealed that the inhibition was caused by an elevation of NADP+ in the incubations and that the incorporation of a NADPH generating system abolished the inhibition. Therefore, neither flavins nor Co-enzyme Q directly affected the 4-ene-steroid 5 alpha-reductase activity. Further evidence to support this conclusion was obtained when several inhibitors of electron transfer reactions failed to inhibit 4-ene-steroid 5 alpha-reductases from rat epididymides, prostate and seminal vesicles. These findings show that, in male rat androgen target tissues, the conversion of testosterone to 5 alpha-dihydrotestosterone does not require intermediates of electron transfer reactions. We propose that the reduction proceeds by the direct transfer of protons from NADPH to testosterone.
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PMID:Mechanism of 4-ene-steroid 5 alpha-reductase proton transfer in androgen target tissues. 674 43

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.
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PMID:Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli. 674 95

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP+ reductase, in a competitive manner with respect to NADPH. The Ki values were 1.5 and 0.2 microM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP+ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP+ reductase than Cibacron blue. The Kd values were 1.9 and 5 microM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP+ oxidoreductase from spinach leaves with good yields.
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PMID:Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography. 682 90

Histochemical studies have been made of the isocitrate dehydrogenase, succinic dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, DPN diaphorase, TPN diaphorase, delta 5-3 beta-hydroxysteroid dehydrogenase and monoamine oxidase in the caput, corpus and cauda epididymides of normal and alpha chlorohydrin (6.5 mg/kg/9 days) treated rats. Administration of alpha chlorohydrin in a low dose caused a conspicuous decrease in all these enzymes except delta 5-3 beta-HSD, in various cell types of epididymal epithelium and sperms. Biochemical estimations of isocitrate dehydrogenase, succinic dehydrogenase, malate dehydrogenase and delta 5-3 beta-HSD have further supported and confirmed these histochemical observations. These changes in enzyme activities after treatment with low dose of alpha chlorohydrin strongly suggest that TCA cycle and amino acid metabolism of epididymis become defective, much earlier before any histological damage to the epididymis becomes visible.
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PMID:Effects of low doses of alpha chlorohydrin on the dehydrogenases and oxidases of rat epididymal epithelium and sperms: a correlative histochemical and biochemical study. 694 44

A systematic investigation of potential ligands for the affinity purification of aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2.) has been carried out. The most suitable nucleotide ligands tested were NADP+ and 2',5'-ADP. Adsorbed enzyme could be eluted with NADPH but not NADH. The chlorotriazinyl dyes Cibacron Blue F3GA and Procion Red HE3B also proved effective as 'affinity' ligands when immobilized to Sepharose 4B. The free dyes and also Blue Dextran (Cibacron Blue F3GA coupled to dextran) were all potent inhibitors of aldehyde reductase. The inhibition by Blue Dextran was shown to be competitive with respect to NADPH (Ki = 1.8 x 10(-7) M). The enzyme was sensitive to inhibition by glutaric acid derivatives, flavonoids and a range of anti-convulsants.
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PMID:Isolation and characterization of rat liver aldehyde reductase. 744 91

The morphology and function of the apical mitochondria-rich cells in the mammalian ductus epididymidis epithelium are revised. These cells are similar in all mammalian species studied. Apical mitochondria-rich cells are scarce (1-5 cells/100 principal cells) and are mainly found in the initial epididymal segments. Their morphology varies from slender cells that extend from the basal lamina to the epididymal lumen, to round cells that protrude into the lumen and are not in contact with the basal lamina. Their cytoplasm is more electron-dense than that of principal cells and contains more mitochondria which, in some species, are surrounded by rough endoplasmic reticulum cisternae. The adluminal cytoplasm displays a few short microvilli and contains many acid phosphatase positive vesicles. Apical mitochondria-rich cells differ from the principal cells in some histochemical features such as: (a) different lectin-staining pattern; (b) more intense reaction to the enzymatic activities: carbonic anhydrase, Ca(2+)-ATPase, peanut-agglutinin-sialidase, NADP dehydrogenase, succinate dehydrogenase, alpha-galactosidase and beta-galactosidase; (c) more intense immunoreaction to several cytokeratin types and to estradiol-related receptor protein; (d) weaker immunoreaction to epithelial membrane antigen and to retinol-binding protein. Although the function of the apical mitochondria-rich cells is still unknown, the following possible functions have been suggested: holocrine secretion; cooperation with the principal cells in epididymal reabsorption of testicular fluid; and acidification of epididymal fluid. Experimental results suggest that differentiation and maintenance of apical mitochondria-rich cells are not under androgen control and that these cells are sensitive to estrogen stimulation.
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PMID:The apical mitochondria-rich cells of the mammalian epididymis. 748 29

The synthesis of NAD and NADP by rat adipose tissue was measured in vitro. Nicotinamide-7-(14)C and NaH(2)(32)PO(4) were incorporated together into NAD with a (32)P/(14)C ratio of 1.82 and nicotinic-7-(14)C acid and NaH(2)(32)PO(4) with a ratio of 1.94. Nicotinic acid stimulated, by 90%, lipogenesis from glucose-U-(14)C by rat adipose tissue in vitro. Glucose plus insulin and refeeding for 48 hr after a 48 hr fast markedly increased the incorporation of nicotinic-7-(14)C into NAD in rat epididymal fat pads in vitro, but neither fructose, L-glutamine, nor insulin alone increased the synthesis of NAD in this tissue. Glucose-1-(14)C, ribose-1-(14)C, and to a greater extent glucose-6-(14)C are incorporated into the NAD of rat adipose tissue. Fasting followed by refeeding sharply increased the radioactivity of NAD-(14)C formed from glucose-1-(14)C and glucose-6-(14)C but not from ribose-1-(14)C. Increasing the ribose concentration from 2 mM to 10 mM increased its incorporation into adipose tissue NAD twofold. The nicotinic-7-(14)C acid incorporation into NAD increased over the 1st hr of incubation and remained constant for the next 3 hr. The concentration of NAD in the fat pads showed a similar response to the time of incubation. NADP concentrations increased over the entire 4 hr incubation period as did the incorporation of nicotinic-7-(14)C acid into NADP. The results of this study suggest that NAD is synthesized de novo by rat adipose tissue in vitro and that this synthesis is increased by factors which stimulate lipogenesis.
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PMID:Pyridine nucleotide synthesis by rat adipose tissue in vitro. 1456 2

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.
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PMID:Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism. 1525 34

Glucose metabolism is necessary for successful fertilization in the mouse. Both spermatozoa and oocytes metabolize glucose through the pentose phosphate pathway (PPP), and NADPH appears required for gamete fusion. The aims of this study were to further characterize the utilization of glucose by the fertilizing spermatozoon and the fertilized oocyte, to demonstrate the importance of the PPP in different steps of fertilization, and to examine whether the beneficial effect of glucose could be mediated by a NADPH-dependent enzyme involved in redox regulation. By using a fluorescent analog of 2-deoxyglucose, glucose uptake was evidenced in both the head and flagellum of motile spermatozoa. After sperm-oocyte fusion, an increase in glucose uptake by the fertilized oocyte was observed but not before the formation of the male and female pronuclei. By using a microphotometric technique, activity of glucose 6-phosphate dehydrogenase (G6PDH), the key enzyme of the PPP, was localized to the sperm head and midpiece. When epididymal spermatozoa were released into a glucose-containing medium, the NADPH/NADP ratio increased with capacitation. Sperm-oocyte fusion and meiosis reinitiation of the fertilized oocyte was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN); inhibition of sperm-oocyte fusion was relieved by NADPH. Sperm-oocyte fusion and meiosis reinitiation were also inhibited by diphenylamine iodonium, which is a flavoenzyme inhibitor reported to prevent reactive oxygen species (ROS) generation in mouse spermatozoa and embryos. These findings indicate that the PPP is involved in different steps of fertilization. Subsequent regulation of a NADPH-dependent flavoenzyme responsible of ROS production is envisaged.
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PMID:Involvement of the pentose phosphate pathway and redox regulation in fertilization in the mouse. 1568 28

11beta-hydroxysteroid dehydrogenase type 1, expressed mainly in the endoplasmic reticulum of adipocytes and hepatocytes, plays an important role in the prereceptorial activation of glucocorticoids. In liver endoplasmic reticulum-derived microsomal vesicles, nicotinamide adenine dinucleotide phosphate reduced supply to the enzyme is guaranteed by a tight functional connection with hexose-6-phosphate dehydrogenase and the glucose-6-phosphate transporter (G6PT). In adipose tissue, the proteins and their activities supporting the action of 11beta-hydroxysteroid dehydrogenase type 1 have not been explored yet. Here we report the occurrence of the hexose-6-phosphate dehydrogenase in rat epididymal fat, as detected at the level of mRNA, protein, and activity. In the isolated microsomes, the activity was evident only on the permeabilization of the membrane because of the poor permeability to the cofactor nicotinamide adenine dineucleotide phosphate (NADP(+)), which is consistent with the intralumenal compartmentation of both the enzyme and a pool of pyridine nucleotides. In fat cells, the access of the substrate, glucose-6-phosphate to the intralumenal hexose-6-phosphate dehydrogenase appeared to be mediated by the liver-type G6PT. In fact, the G6PT expression was revealed at the level of mRNA and protein. Accordingly, the transport of glucose-6-phosphate was demonstrated in microsomal vesicles, and it was inhibited by S3483, a prototypic inhibitor of G6PT. Furthermore, isolated adipocytes produced cortisol on addition of cortisone, and the production was markedly inhibited by S3483. The results show that adipocytes are equipped with a functional G6PT-hexose-6-phosphate dehydrogenase-11beta-hydroxysteroid dehydrogenase type 1 system and indicate that all three components are potential pharmacological targets for modulating local glucocorticoid activation.
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PMID:The glucose-6-phosphate transporter-hexose-6-phosphate dehydrogenase-11beta-hydroxysteroid dehydrogenase type 1 system of the adipose tissue. 1730 57


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