Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been found that metyrapone can inhibit both type I and type II mixed-function oxygenase reactions, while cysteamine inhibits only type I activity in this mammalian system. Following pretreatment with phenobarbital and 3-methylcholanthrene the half-maximal inhibiting concentrations for the O-demethylation of paranitranisol are increased for cysteamine and decreased for metyrapone. Both cysteamine and metyrapone give type II binding spectra with oxidized cytochrome P-450. The negative and positive peaks are at 393 and 426 nm respectively for metyrapone, and 410 and 434 nm for cysteamine. Cysteamine showed no binding comparable to that of metyrapone for reduced cytochrome P-450. Metyrapone showed little or no inhibition of the NADH cytochrome-c reductase (EC 1.6.1.1) or NADPH (EC 1.6.2.3) cytochrome-c reductase while cysteamine had a more or less strong inhibiting effect depending on the pretreatment of animals. Neither the binding to P-450 heme nor the inhibition of NADH and NADPH cytochrome-c reductase correlates well with cysteamine inhibition of total activity. It is therefore suggested that cysteamine reacts with an intermediate electron carrier of non-heme iron or glycoprotein character thus inhibiting mixed-function oxygenase activity.
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PMID:A comparative study on the influence of cysteamine and metyrapone on mixed-function oxygenase activities in variously pretreated liver microsomes from rats and mice. 13 29

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.
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PMID:Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum. 137 17

It has been suggested by a number of investigators that glycoproteins may play a role in the development and/or maintenance of synapses in the mammalian CNS. For many synaptic glycoproteins, however, little precise structural or functional information is available. In an effort to isolate probes specific to individual glycoproteins, we have screened a rat brain cDNA expression library with a mixed polyclonal antibody directed against concanavalin A-binding synaptic junctional glycoproteins. Using this approach, we have previously reported the cloning of SC1, a putative extracellular matrix glycoprotein found in adult brain (Johnston et al., Neuron 2:165-176, 1990). We now report the cloning and characterization of a second novel cDNA, which has been designated SC2. Northern blots show that this cDNA recognizes a 1.2-kb mRNA that is present throughout postnatal development in the rat. It is expressed at high levels in brain and is also found at lower levels in several other tissues. In situ hybridization suggests that the SC2 mRNA is strongly expressed by many types of neurons. Sequence data reveals a single open reading frame in the cDNA, encoding a putative hydrophobic protein with a calculated molecular weight of 36.1 kDa. Sequence analysis reveals some similarity between SC2 and 5 alpha-reductase, a microsomal membrane protein important in testosterone metabolism.
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PMID:Molecular cloning of a novel mRNA using an antibody directed against synaptic glycoproteins. 140 91

The Na(+)-H+ antiport is a membrane-bound glycoprotein that extrudes intracellular acid loads and regulates cellular volume. Cellular synthesis of the oligosaccharide side chains of glycoproteins is dependent on a supply of mevalonate, itself a product of the rate-limiting enzyme of cholesterol synthesis 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The effect of two HMG CoA reductase inhibitors (simvastatin and 25-hydroxycholesterol) on intracellular pH and Na(+)-H+ exchange was therefore studied. Inhibition of the Na(+)-H+ antiport by these agents led to a fall in intracellular pH but did not impair the regulatory volume increase response to a hypertonic stimulus. The inhibitory effect of simvastatin was prevented by mevalonate but not dolichol or squalene. The effect of 25-hydroxycholesterol was more complex and not easily reversed. Thus HMG CoA reductase inhibitors reduced the ability of human lymphoblasts to expel an intracellular acid load via the Na(+)-H+ antiport, although the response of the antiport to an osmotic stimulus was preserved.
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PMID:HMG CoA reductase inhibitors affect Na(+)-H+ antiport activity in human lymphoblasts. 165 6

The phenotypic expression of multidrug resistance by the doxorubicin-selected AdrR human breast tumor cell line is associated with overexpression of plasma membrane P-170 glycoprotein and increased cytosolic selenium-dependent GSH-peroxidase activity relative to the parental MCF-7 wild-type line (WT). To determine whether doxorubicin resistance by AdrR cells persists in vivo, and to further investigate the possibility of biochemical differences between WT and AdrR solid tumors, both tumor cell lines were grown as subcutaneous xenografts in athymic nude mice. Tumorigenicity depended upon cell inoculation burden, and tumor incidence was similar for both cell lines (greater than 80% tumor takes at 10(7) cells/mouse) at 14 days, provided 17 beta-estradiol was supplied to the animals bearing the WT tumors. However, the growth rate for the AdrR xenografts was only about half that of WT xenografts. Doxorubicin (2-8 mg/kg, i.p., injected weekly) significantly diminished the growth of the WT tumors, but AdrR solid tumors failed to respond to doxorubicin. The accumulation of 14C-labeled doxorubicin was 2-fold greater in WT xenografts that in AdrR, although there were no differences in host organ drug levels in mice bearing either type of tumors. Membrane P-170 glycoprotein mRNA was detected by slot-blot analysis in the AdrR tumors, but not in WT. Electron spin resonance 5,5-dimethylpyrroline-N-oxide-spin-trapping experiments with microsomes and mitochondria from WT and AdrR xenographs demonstrated a 2-fold greater oxygen radical (superoxide and hydroxyl) formation from activated doxorubicin with WT xenographs compared to AdrR. Selenium-dependent glutathione (GSH)-peroxidase, superoxide dismutase and GSH-S-aryltransferase activities in AdrR xenografts were elevated relative to WT. Although the activities of the latter two enzymes were similar to those measured in both tumor cell lines, GSH-peroxidase activities were elevated 70-fold (WT) and 10-fold (AdrR) in xenografts compared to tumor cells. In contrast, in both WT and AdrR solid tumors in vivo, catalase, NAD(P)H-oxidoreductases, and glutathione disulfide (GSSG)-reductase activities, and GSH and GSSG levels were not markedly different, and were essentially the same as in cells in vitro. Like the MDR cells in culture, AdrR tumor xenografts were extremely resistant to doxorubicin and retained most of the characteristics of the altered phenotype. These results suggest that WT and AdrR breast tumor xenografts provide a useful model for the study of biochemical and pharmacological mechanisms of drug resistance by solid tumors in vivo.
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PMID:Biochemical and pharmacological characterization of MCF-7 drug-sensitive and AdrR multidrug-resistant human breast tumor xenografts in athymic nude mice. 167 69

The immunoprecipitation by antibodies to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of extracts of [35S]methionine-pulse-labelled isolated hepatocytes, followed by electrophoresis and fluorography, showed the presence not only of 97-kDa HMG-CoA reductase, but also of another protein of 180 kDa. Boiling the immunoprecipitates both in the presence and in the absence of 2-mercaptoethanol, followed by SDS/polyacrylamide gel electrophoresis both in the presence and in the absence of 8 M urea, was not found to change the ratio of 180-kDa/97-kDa proteins. These facts suggest that the 180-kDa protein is not an aggregated form of HMG-CoA reductase. A different batch of antibodies obtained from a newly purified HMG-CoA reductase fully titrated the reductase activity, but did not immunoprecipitate the 180-kDa protein, showing that there is no cross-reactivity between these proteins. The 180-kDa polypeptide is a glycoprotein of N-linked high-mannose oligosaccharide chains, which is not processed on the Golgi system. The apparent molecular mass of the carbohydrate is 16 kDa. The incubation of rat hepatocytes with sterols produces, on the one hand, a decrease in the rate of synthesis, and on the other hand, an acceleration in the turnover rate of the 180-kDa protein. In addition, mevalonate is known to decrease its rate of synthesis. The carbohydrate-free 164-kDa protein was found to degrade only a tenth as fast as the glycoprotein and, furthermore, the degradation was no longer accelerated by sterols. These results support the notion that the 180-kDa protein is not a modified form of 97-kDa reductase, but probably a different protein related to cholesterol metabolism, and also that the N-linked, high-mannose chains, which are bound to the glycoprotein, are required for rapid and controlled degradation of the protein.
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PMID:Identification of a cholesterol-regulated 180-kDa microsomal protein in rat hepatocytes. 196 43

In a serum-free medium we have established two new human breast carcinoma cell lines from a single primary tumor. Cultures were maintained on chemically defined medium CDM3 or on minor modifications of this medium, Dulbecco's modified Eagle medium-Ham's F12 supplemented with epidermal growth factor, insulin, transferrin, estradiol, hydrocortisone, triiodothyronine, cyclic AMP, phosphoethanolamine, ethanolamine, fibronectin, fetuin, ascorbic acid, bovine serum albumin, and trace element salts including selenite (Petersen and van Deurs, Cancer Res., 47: 856-866, 1987). Primary cultures comprised both NADPH-neotetrazolium reductase-positive carcinoma cells and NADPH-neotetrazolium reductase-negative cells of stromal appearance, as well as normal epithelial cells (Petersen and van Deurs, Cancer Res., 46: 2013-2020, 1986). In subsequent passages the cells were monitored exclusively using the tumorigenicity assay on nude mice. Two cell lines, one nontumorigenic, HMT-3909S1, and one tumorigenic, HMT-3909S8, were selected from the primary cultures. Selection of S8 through subline S4 required transient supplementation of CDM3 with fetal calf serum. Permanent lines S1 and S8 were maintained on serum-free medium. Further characterization of the two cell lines in terms of normal breast gland differentiation (Petersen and van Deurs, Differentiation, 39: 197-215, 1988) was carried out using immunocytochemistry, immunochemistry, electron microscopy, and cytogenetics. S8 appeared to be identical with the NADPH-neotetrazolium reductase-positive carcinoma cells of the primary cultures, with a particular subpopulation of carcinoma cells in the tumor of origin, and with the tumorigenic cells of the nude mice. This subline was aneuploid, typically epithelial in morphology, and expressed keratins K8 and K18 and the glycoprotein MAM-6, typical of luminal epithelial cells in the normal breast gland. Subline S1 appeared more like the elongated cells in the primary cultures and like a second subpopulation of cells in the carcinoma of origin. However, S1 cells were in fact epithelial, since they expressed keratins. Also, S1 cells seemed to be a triploidation of a cell with close resemblance to S4, while only few cytogenetic differences were found between S4 and S8, suggesting an origin of S1 and S8 via S4 from a single hypothetical stem cell.
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PMID:Differential tumorigenicity of two autologous human breast carcinoma cell lines, HMT-3909S1 and HMT-3909S8, established in serum-free medium. 215 55

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the limiting enzyme step in cholesterol formation in mammalian liver and other tissues. It is a glycoprotein of 97,000 daltons embedded in the endoplasmic reticulum with a long cytoplasmic extension that is the site of catalytic conversion of HMG CoA to mevalonate. The enzyme is subject to both long-term (induction/repression; degradation) and short-term control (reversible phosphorylation) mediated by endocrine signaling (insulin, glucagon) and through negative feedback by metabolic products of mevalonate (e.g., cholesterol). The catalytic capacity of microsomal reductase falls rapidly in the presence of several protein kinases (reductase kinase, protein kinase-C, calmodulin-dependent protein kinase). Activity is restored with various protein phosphatases. Increased phosphorylation of reductase in intact cells after addition of glucagon or mevalonate is followed by enhanced degradation of the enzyme. In an in vitro model system, phosphorylated, native microsomal reductase is more rapidly cleaved by the calcium-dependent, neutral protease calpain than the dephosphorylated from of reductase. Our present research which centers on the mechanism of the in vitro model system is reviewed. Calpain in the presence of Ca2+ cleaves the cytosolic domain of phosphorylated 97 kDa reductase at two points giving rise to two fragments of nearly the same size that appear as a 52-56,000 dalton doublet by electrophoresis and immunoblotting. In the same system native reductase labeled with [gamma-32P]ATP generates a doublet with 32P solely in the upper (heavier) band. This indicates that serine phosphorylation sites lie between the two calpain cleavage loci. These are positioned in the "linker" region of the long carboxy-terminal cytosolic domain near the membrane. This segment possesses five invariant serine residues and two PEST sequences (constellations of proline, glutamate, serine and threonine) that are characteristic of proteins with short half-lives. If phosphorylation of HMG CoA reductase is confined to the linker region, we must look to this domain in order to interpret the resulting conformational changes that markedly influence reductase catalytic activity and prepare the enzyme for degradation.
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PMID:Phosphorylation and degradation of HMG CoA reductase. 262 76

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.
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PMID:The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells. 293 79

A full length cDNA for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, the membrane-bound glycoprotein that regulates cholesterol synthesis, was isolated from a human fetal adrenal cDNA library. The nucleotide sequence of this cDNA shows that the human reductase is 888 amino acids long and shares a high degree of homology with the hamster enzyme. The amino-terminal membrane-bound domain is the most conserved region between the two species (7 substitutions out of 339 amino acids). This region, which is predicted to span the endoplasmic reticulum membrane seven times, mediates accelerated degradation of reductase in the presence of sterols. The carboxyl-terminal catalytic domain is also highly conserved (22 substitutions out of 439 amino acids). However, the linker region between these two domains has diverged (32 substitutions out of 110 amino acids). Conservation of the structure of the membrane-bound domain in HMG-CoA reductase supports the hypothesis that sterol-regulated degradation is an important mechanism for suppression of reductase activity and for regulation of cholesterol metabolism in humans as well as in hamsters.
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PMID:Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation. 299 Dec 81


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