Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Spin-labeled copolymers of 4-thiouridine and uridine (ls4U,U)n] that contain various amounts of spin label (l) were synthesized by either (i) chemical alkylation of the 4-thiouridine-uridine copolymers (s4U,U)n prepared by copolymerizing 4-thiouridine 5'-diphosphate (s4UDP) and UDP or (ii) copolymerization of spin-labeled s4UDP with UDP using polynucleotide phosphorylase. The effect of (s4U,U)n and (ls4U,U)n on avian myeloblastosis virus (AMV) RNA-dependent DNA polymerase (RNA-dependent DNA nucleotidyltransferase, EC 2.7.7.7; reverse transcriptase) was studied to determine whether the presence of potentially reactive thiol groups or spin labels enhances the inhibitory properties of the copolymers as compared to (U)n. Inhibition by (s4U,U)n gradually increases as the percentage of thiolation increases. Enhanced inhibition by (s4U,U)n appears to be due to the interaction of the thiol groups of (s4U,U)n with the thiol group(s) of the polymerase, because inhibition by (s4U,U)n (8% thiolated) in the presence of dithiotreitol resembles that by (U)n. In contrast, inhibition by (ls4U,U)n containing 3% spin label resembles that by (U)n; however, increasing the spin label to 6% or 12% results in enhanced inhibition by (ls4U,U)n as compared to that by (U)n, and dithiothreitol has no effect on enhanced inhibition by (ls4U,U)n. These results suggest that the mechanism of inhibition observed with (ls4U,U)n with a ls4U:U ratio > 1:33 differs from the mechanism for (s4U,U)n and involves complex formation between the spin label and the essential Zn2+ of RNA-dependent DNA polymerase.
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PMID:Reactivity of reverse transcriptase toward (s4U,U)n copolymers and spin-labeled nucleic acid lattices. 615 32

The cDNA clone encoding human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-1 (GalNAc-T1) was isolated from colon tissue by a reverse transcriptase-polymerase chain reaction (RT-PCR). Using fluorescence in situ hybridization, the position of the GalNAc-T1 gene was shown to be localized at the human chromosomal region, 18q12.1.
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PMID:A human UDP-GalNAc: polypeptide, N-acetylgalactosaminyltransferase type 1 gene is located at the chromosomal region 18q12.1. 905 Sep 10

Genetic polymorphisms occur in many of the drug metabolizing enzymes. However, the effect of polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases is still undescribed, despite the many reported cases of variations in glucuronidation activities. Characterization of the UGT2B15(Y85) cDNA, which was isolated from human prostate and LNCaP cell cDNA libraries, revealed 20 nucleotide differences between UGT2B15(Y85) and the previously characterized UGT2B15 protein UGT2B15(D85). However, only one of the two variations in the coding region leads to an amino acid change from aspartic acid to a tyrosine residue at position 85. The genomic DNA of 27 subjects were analysed by direct sequencing of polymerase chain reaction (PCR) products and demonstrated that UGT2B15(D85) and UGT2B15(Y85) are encoded by variant alleles prevalent in the Caucasian population. Expression of UGT2B15(D85) and UGT2B15(Y85) in HK293 cells demonstrated similar substrate specificities. Of the 65 potential substrates tested for activity, the proteins were active on phenolic compounds, coumarins, flavonoids, drugs and steroid hormones. Both proteins displayed similar Km values of 2.2 and 2.4 microM for androstane-3alpha,17beta-diol and dihydrotestosterone, respectively. However, results suggest that UGT2B15(Y85) has a higher Vmax than UGT2B15(D85). Specific reverse transcriptase (RT)-PCR analysis revealed expression of the UGT2B15 gene in a wide range of extrahepatic tissues including the human liver, kidney, testis, mammary gland, placenta, adipose, skin, uterus, prostate and lung. The wide expression of UGT2B15 in many tissues indicates that it is a major glucuronidation enzyme in humans.
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PMID:Isolation and characterization of UGT2B15(Y85): a UDP-glucuronosyltransferase encoded by a polymorphic gene. 929 60

1. Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. 3. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. 4. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 5. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6. Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. 7. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]-thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. 8 These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.
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PMID:Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP. 969 Aug 62

Human UDP-glucuronosyltransferases (UGTs) are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues [Strassburg, Manns and Tukey (1998) J. Biol. Chem. 273, 8719-8726]. Previous work suggests that these enzymes play a protective role in chemical carcinogenesis [Strassburg, Manns and Tukey (1997) Cancer Res. 57, 2979-2985]. In this study, UGT1 and UGT2 gene expression was investigated in human oesophageal epithelium and squamous-cell carcinoma in addition to the characterization of individual UGT isoforms using recombinant protein. UGT mRNA expression was characterized by duplex reverse transcriptase-PCR analysis and revealed the expression of UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mRNAs. UGT1A1, UGT1A3, UGT1A4, UGT1A5 and UGT1A6 transcripts were not detected. UGT2 expression included UGT2B7, UGT2B10 and UGT2B15, but UGT2B4 mRNA was absent. UGT2 mRNA was present at significantly lower levels than UGT1 transcripts. This observation was in agreement with the analysis of catalytic activities in oesophageal microsomal protein, which was characterized by high glucuronidation rates for phenolic xenobiotics, all of which are classical UGT1 substrates. Whereas UGT1A9 was not regulated, differential regulation of UGT1A7 and UGT1A10 mRNA was observed between normal oesophageal epithelium and squamous-cell carcinoma. Expression and analysis in vitro of recombinant UGT1A7, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 demonstrated that UGT1A7, UGT1A9 and UGT1A10 catalysed the glucuronidation of 7-hydroxybenzo(alpha)pyrene, as well as other environmental carcinogens, such as 2-hydroxyamino-1-methyl-6-phenylimidazo-(4, 5-beta)-pyridine. Although UGT1A9 was not regulated in the carcinoma tissue, the five-fold reduction in 7-hydroxybenzo(alpha)pyrene glucuronidation could be attributed to regulation of UGT1A7 and UGT1A10. These data elucidate an individual regulation of human UGT1A and UGT2B genes in human oesophagus and provide evidence for specific catalytic activities of individual human UGT isoforms towards environmental carcinogens that have been implicated in cellular carcinogenesis.
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PMID:Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus. 1002 27

Although enzymatic processes involved in the formation of active steroids are well known, less information is available about the enzymes responsible for the metabolism of these hormones. Moreover, the expression of these catabolic enzymes, which include UDP-glucuronosyltransferases, may play a role in the regulation of the level and action of steroid hormones in steroid target tissues. Previous studies have shown that the cynomolgus monkey contains high levels of circulating androgen glucuronides, indicating that it represents the best animal model to study the glucuronidation of steroids in extrahepatic tissues. Two cDNA libraries were constructed from monkey liver and prostate mRNA, and a novel UDP-glucuronosyltransferase UGT2B cDNA, UGT2B19, was isolated from both libraries. The UGT2B19 cDNA is 2108 bp in length and contains an open reading frame of 1584 bp encoding a protein of 528 residues. The UGT2B19 cDNA clone was transfected into HK293 cells and a stable cell line expressing UGT2B19 protein was established. The activity of UGT2B19 on 3alpha-hydroxy and 17beta-hydroxy positions of steroids was demonstrated. The enzyme also conjugates xenobiotics including eugenol, 1-naphthol and p-nitrophenol. Kinetic analysis revealed that UGT2B19 glucuronidates steroids with Km values of 1.6, 2.6 and 4.3 microm for testosterone, etiocholanolone and 5beta-androstane-3alpha,17beta-diol, respectively. UGT2B19 transcript was detected, by specific reverse transcriptase-PCR analysis in the liver, ovary, prostate, colon, spleen, kidney, pancreas, brain, cerebellum, mammary gland and epididymis. The molecular characterization of simian UGT2B19 demonstrates relevance of using monkey as an animal model to study and understand steroid glucuronidation in extrahepatic target tissue.
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PMID:Molecular cloning, expression and characterization of a monkey steroid UDP-glucuronosyltransferase, UGT2B19, that conjugates testosterone. 1010 98

Variations in glucuronidation activities among different individuals have been reported; however, genetic polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases have not been studied extensively. A novel UGT2B cDNA clone UGT2B4(E458) was isolated from human prostate and LNCaP cell cDNA libraries. The cDNA encoding UGT2B4(E458) is 2097 bp in length and has an open reading frame of 1584 nucleotides encoding a protein of 528 amino acids. Characterization of the UGT2B4(E458) cDNA revealed nucleotide differences with the previously published UGT2B4 and UGT2B11 cDNAs. These variations in the UGT2B4 sequence lead to an amino acid change from aspartic acid to glutamic acid at position 458. In the previous UGT2B11 cDNA (which has subsequently been renamed UGT2B4 (L109,396, D458)), leucine residues are found at positions 109 and 396, whereas phenylalanines are present at these positions in the UGT2B4(D458) and UGT2B4(E458) enzymes. Analysing the genomic DNA of 26 unrelated Caucasian individuals demonstrated the presence of variant alleles encoding UGT2B4(D458) and UGT2B4(E458). Stable expression of UGT2B4(E458) cDNA in HK293 cells demonstrates the presence of a 52 kDa protein, which is in agreement with other characterized (UGT2B proteins. UGT2B4(E458) conjugates hyodeoxycholic acid (HDCA) as well as 4-hydroxyestrone (4-OH-E1), androstane-3alpha,17beta-diol (3alpha-diol) and androsterone (ADT). Specific reverse transcriptase-polymerase chain reaction analysis revealed expression of UGT2B4(D458) and UGT2B4(E458) transcripts in a wide range of extrahepatic tissues, including the liver, kidney, testis, mammary gland, prostate, placenta, adipose, adrenal, skin and lung. Our results suggest that UGT2B4(E458) and UGT2B(E458) are two widely expressed isoenzymes, and that polymorphism in the UGT2B4 gene might be responsible for differences in UGT2B4 enzymatic properties.
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PMID:Characterization and substrate specificity of UGT2B4 (E458): a UDP-glucuronosyltransferase encoded by a polymorphic gene. 1037 68

Sulfolipids of photosynthetic bacteria and plants are characterized by their unique sulfoquinovose headgroup, a derivative of glucose in which the 6-hydroxyl group is replaced by a sulfonate group. These sulfolipids have been discussed as promising anti-tumor and anti-HIV therapeutics based on their inhibition of DNA polymerase and reverse transcriptase. To study sulfolipid biosynthesis, in particular the formation of UDP-sulfoquinovose, we have combined computational modeling with biochemical methods. A database search was performed employing the derived amino acid sequence from SQD1, a gene involved in sulfolipid biosynthesis of Arabidopsis thaliana. This sequence shows high similarity to other sulfolipid biosynthetic proteins of different organisms and also to sugar nucleotide modifying enzymes, including UDP-glucose epimerase and dTDP-glucose dehydratase. Additional biochemical data on the purified SQD1 protein suggest that it is involved in the formation of UDP-sulfoquinovose, the first step of sulfolipid biosynthesis. To understand which aspects of epimerase catalysis may be shared by SQD1, we built a three-dimensional model of SQD1 using the 1.8 A crystallographic structure of UDP-glucose 4-epimerase as a template. This model predicted an NAD(+) binding site, and the binding of NAD(+) was subsequently confirmed by enzymatic assay and mass spectrometry. The active-site interactions together with biochemical data provide the basis for proposing a reaction mechanism for UDP-sulfoquinovose formation.
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PMID:Prediction of the active-site structure and NAD(+) binding in SQD1, a protein essential for sulfolipid biosynthesis in Arabidopsis. 1046 38

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.
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PMID:Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors. 1068 1

Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC 2.4.2.26, XT-I) initiates the biosynthesis of glycosaminoglycan chains in proteoglycans by transferring xylose from UDP-xylose to specific serine residues of the core protein. Based on the partial amino acid sequence of the purified enzyme from human JAR choriocarcinoma cell culture supernatant we isolated a cDNA encoding XT-I using the degenerate reverse transcriptase-polymerase chain reaction method. This enzyme, which is involved in chondroitin sulfate, heparan sulfate, heparin and dermatan sulfate biosynthesis, belongs to a novel family of glycosyltransferases with no homology to proteins known so far. 5' and 3'-RACE were performed to isolate a novel cDNA fragment of 3726 bp with a single open reading frame encoding at least 827 amino acid residues with a molecular mass of 91 kDa. The human XT-I gene was located on chromosome 16p13.1 using radiation hybrid mapping, and extracts from CHO-K1 cells transfected with the XT-I cDNA in an expression vector exhibited marked XT activity. A new 3608 bp cDNA fragment encoding a protein of 865 amino acid residues was also isolated by PCR using degenerate primers based on the amino acid sequence of human XT-I. The amino acid sequence of this XT-II isoform displayed 55% identity to the human XT-I. The XT-II gene was located on chromosome 17q21.3-17q22, and the exon/intron structure of the 15 kb gene was determined. RT-PCR analyses of XT-I and XT-II mRNA from various tissues confirmed that both XT-I and XT-II transcripts are ubiquitously expressed in the human tissues, although with different levels of transcription. Furthermore, the cDNAs encoding XT-I and XT-II from rat were cloned. The deduced amino acid sequences of rat xylosyltransferases displayed 94% identity to the corresponding human enzyme.
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PMID:Molecular cloning and expression of human UDP-d-Xylose:proteoglycan core protein beta-d-xylosyltransferase and its first isoform XT-II. 1109 77


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