Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously demonstrated fucosyltransferase (FT) activity on mouse germ cell surfaces at different stages of spermatogenesis. To complement these findings, here we report FT activity on the Sertoli cell (SC) surface. SC isolated and cultured from 20-day-old rat testes displayed FT activity with a Vmax of 12.5 pmoles/mg protein/min and a Km of 22 microM, while purified Sertoli cell plasma membranes (SCPM) showed FT activity with a Vmax of 10 pmoles/mg protein/min and a Km of 18.2 microM for GDP-[14C]-L-fucose. Fucosyltransferase activities were 16.7 and 2.6 pmoles/mg protein/min in SC and SCPM, respectively; approximately 16% of FT activity is, therefore, on the cell surface. To test whether the expression of FT activity in SC was regulated by hormones and growth factors, SC were cultured in serum-free medium supplemented with insulin, transferrin, sodium selenite, and epidermal growth factor (medium 4F) or in 4F plus follicle-stimulating hormone, testosterone, hydrocortisone, and vitamin E (medium 8F). We found that FT activity in SC is not modulated by these hormones or growth factors (4F or 8F). For comparison with FT, galactosyltransferase (GalTase) activities in SC and SCPM were also determined. SC displayed GalTase activity with a Vmax of 50 pmoles/mg protein/min and a Km of 38.5 microM, while SCPM showed GalTase activity with a Vmax of 25 pmoles/mg protein/min and a Km of 20.8 microM for UDP-[3H]-galactose. Galactosyl-transferase activities were 29.2 and 9.6 pmoles/mg protein/min in SC and SCPM, respectively. Therefore, approximately 33% of the total cell GalTase activity was detected on the surface membranes of rat Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1993 Oct
PMID:Surface-associated glycosyltransferase activities in rat Sertoli cells in vitro. 825 68

The possibility that the firA gene of Escherichia coli (Dicker, I. B., and Seetharam, S. (1991) Mol. Microbiol. 6, 817-823) might function in lipid A biosynthesis was examined based on its homology to the lpxA gene, which encodes UDP-N-acetylglucosamine O-acyl-transferase, the first enzyme in lipid A formation. Extracts of a temperature-sensitive firA mutant, RL-25, were assayed for their ability to acylate UDP-GlcNAc, using a coupled assay. The results suggested that extracts of RL-25 might be defective in the third enzyme of this pathway, the UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. Living cells of RL-25 also displayed a 5-fold decreased rate of lipid A biosynthesis at the nonpermissive temperature as judged by a 32Pi incorporation assay. In order to examine N-acyltransferase activity directly, the substrate [alpha-32P]UDP-3-O-(R-3-hydroxymyristoyl)-GlcN was synthesized enzymatically. N-Acyltransferase specific activity in RL-25 extracts was reduced to less than 10% of wild-type. When the wild-type firA gene was cloned into a T7-based expression vector, N-acyltransferase specific activity increased almost 360-fold relative to wild-type extracts, demonstrating that firA is the structural gene for the enzyme. The N-acyltransferase displays absolute specificity for the R-3-OH moiety of R-3-hydroxymyristoyl-ACP, as does the O-acyltransferase, consistent with the placement of R-3-hydroxymyristate in E. coli lipid A.
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PMID:The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis. 836 25

The incorporation of radioactive precursors into pyrimidine nucleotides via de novo and salvage pathways was measured in gravid Angiostrongylus cantonensis by HPLC and thin-layer chromatography. 14C-labelled orotate, uridine, uracil and deoxyuridine were traced to UMP, UDP, UTP, UDP-glucose, dTMP, CMP, CDP and CTP. 3H-labelled cytidine was also incorporated into both uracil and cytosine nucleotides in a ratio of 2:1. Cytosine was a major end-product for all the precursors. Cytosine nucleotides were probably formed from UTP by the action of CTP synthetase whose activity in crude cell-free extract was 31.5 +/- 4.9 pmol min-1 (mg protein)-1. It was dependent on glutamine, ATP and GTP and was inhibited by CTP. The total amount of pyrimidine nucleotides formed from uridine was 3 times of that from uracil. The presence of uracil in the metabolism of uridine indicates that UMP is formed by uracil phosphoribosyltransferase as well as by uridine kinase. UMP is a key intermediate for cytidylate and thymidylate biosynthesis in the gravid worms.
Mol Biochem Parasitol 1993 Jul
PMID:Pathways of pyrimidine nucleotide biosynthesis in gravid Angiostrongylus cantonensis. 836 94

To differentiate the effects of GDP and GTP on adenylyl cyclase regulation, adenylyl cyclase in canine sarcolemmal membranes was studied under conditions where only 3-12% of added GDP was converted to GTP by membrane-associated nucleoside diphosphate kinase. Adenylyl cyclase was stimulated up to 180% by GDP at 7-fold lower concentrations than required for stimulation by GTP (GDP half-maximal activation, 120 nM; GTP half-maximal activation, 830 nM). Transphosphorylation of GDP to GTP was blocked completely by the addition of 3 mM UDP. However, UDP did not affect GDP-mediated adenylyl cyclase activation, and guanosine 5'-O-(2-thiodiphosphate) had the same effect on adenylyl cyclase activity as did GDP, indicating that GDP-mediated stimulation of adenylyl cyclase was not due to transphosphorylation of GDP to GTP. Carbachol inhibited GDP-stimulated adenylyl cyclase activity even without addition of GTP; however, this inhibition was clearly dependent upon the endogenous formation of GTP. Half-maximal adenylyl cyclase inhibition by carbachol required the addition of either 330 nM GDP or 25 nM GTP. Taking into account a 3-12% conversion of GDP to GTP by membrane-associated nucleoside diphosphate kinase, sufficient GTP was generated from GDP to support receptor-mediated inhibition of adenylyl cyclase. In addition carbachol-mediated adenylyl cyclase inhibition in the presence of GDP, but not GTP, was blocked completely by 3 mM UDP. In conclusion, GDP-activated adenylyl cyclase could be inhibited by carbachol in the presence of GTP concentrations that were 34-fold below the concentrations needed for GTP-mediated activation of stimulatory guanine nucleotide-binding proteins. In addition, at low GTP concentrations carbachol reduced adenylyl cyclase to levels below "basal" activity (activity in the absence of guanine nucleotides). Although indirectly, these results suggest that carbachol-mediated inhibition of adenylyl cyclase may be independent of Gs activity and possibly due to direct interaction of inhibitory guanine nucleotide-binding proteins and adenylyl cyclase.
Mol Pharmacol 1993 Jan
PMID:Muscarinic receptor-mediated inhibition of GDP-activated adenylyl cyclase suggests a direct interaction of inhibitory guanine nucleotide-binding proteins and adenylyl cyclase. 838 Aug 86

Detection of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of beta-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of beta-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface beta-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of beta-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-beta-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn2+ and UDP-[3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[3H]-galactose, beta-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > alpha chain > mu chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, beta-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the beta-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, alpha-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37 degrees C, the apparent affinity constants and association rate constants of interaction between cell surface beta-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and alpha 2 chain or monomeric IgA1 were in the range from 7.1 x 10(7) to 4.6 x 10(8) M-1 and from 1 x 10(5) to 3 x 10(6) M-1 s-1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 x 10(-2) to 5.0 x 10(-4) s-1 and from 33 to 1380 s, respectively. The number of alpha 2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 x 10(5) to 1.3 x 10(6). The binding of IgA by the cell surface beta-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.
Mol Immunol 1993 Feb
PMID:Interactions of cell-surface galactosyltransferase with immunoglobulins. 843 5

Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Immunol 1993 Feb
PMID:Interactions of galactosyltransferase with serum and secretory immunoglobulins and their component chains. 843 6

The synthetic estrogen ethinylestradiol is extensively eliminated as glucuronide metabolites in humans, but the UDP-glucuronosyltransferases (UGTs) catalyzing this reaction have not been identified. Therefore, ethinylestradiol was tested as a substrate for cloned human UGTs stably expressed in V79 cell lines. Two cloned expressed human enzymes, a bilirubin UGT and a phenol UGT, were observed to catalyze the glucuronidation of ethinylestradiol. High performance liquid chromatographic analysis of the products formed revealed that the expressed bilirubin UGT specifically produced ethinylestradiol-3-glucuronide. In human liver microsomes the ratio of 3-glucuronide/17-glucuronide was 97:3. Subsequent study of the cloned expressed enzymes and human liver microsomes from Crigler-Najjar patients by kinetic analysis and by substrate inhibition strongly indicated that a human liver bilirubin UGT was largely responsible for glucuronidation of ethinylestradiol. These results may provide an explanation for jaundice caused by ethinylestradiol in certain susceptible individuals.
Mol Pharmacol 1993 Apr
PMID:Human bilirubin UDP-glucuronosyltransferase catalyzes the glucuronidation of ethinylestradiol. 847 33

A direct phosphate transfer reaction from the G protein beta subunits to either Gs alpha or Gi alpha has been proposed to account for the ability of thiophosphorylated transducin beta gamma-dimers to bidirectionally regulate adenylyl cyclase activity in human platelet membranes. We searched for experimental evidence for this reaction. Incubation of human platelet membranes with [35S]guanosine-5'-(3-O-thio)triphosphate ([35S]GTP gamma S) results in the predominant incorporation of [35S]thiophosphate into a 36-kDa protein, which comigrates with the G protein beta subunit and is immunoprecipitated by a beta subunit-specific antiserum. Thiophosphorylation of the beta subunit is specific for guanine nucleotides and abolished by the histidine-modifying agent diethylpyrocarbonate and heat and acid treatment. Dephosphorylation of [35S]thiophosphorylated beta subunits is accelerated in the presence of GDP, but not ADP, UDP, or guanosine-5'-(2-O-thio)diphosphate. Neither the thiophosphorylation nor the dephosphorylation is sensitive to receptor agonists (alpha 2-adrenergic, A2 adenosine, thrombin, or insulin), and purified G protein alpha subunits do not act as thiophosphate donors. An approach was designed to demonstrate direct thiophosphate transfer to protein-bound nucleotides; platelet membranes were sequentially exposed to NaIO4, NaCNBH3, and NaBH4, an oxidation-reduction step that covalently incorporates prebound nucleotides into proteins. Under these conditions, multiple radiolabeled proteins are visualized on subsequent addition of [35S]GTP gamma S. This reaction is specific because both oxidation and reduction are required and pretreatment of platelet membranes with 2',3'-dialdehyde GTP gamma S or diethylpyrocarbonate blocks the subsequent labeling in oxidized and reduced membranes. The G protein beta subunit may participate in this thiophosphate transfer reaction. Most important, however, no labeled G protein alpha subunits (Gs alpha and Gi alpha) were recovered by immunoprecipitation from oxidized and reduced membranes subsequent to the addition of [35S]GTP gamma S. Thus, our results clearly rule out the existence of a postulated G protein activation by phosphate transfer reactions, which lead to the formation of GTP from GDP prebound to the alpha subunit.
Mol Pharmacol 1996 Jan
PMID:Thiophosphorylation of the G protein beta subunit in human platelet membranes: evidence against a direct phosphate transfer reaction to G alpha subunits. 856 15

The UDP-glucuronosyltransferase (EC 2.4.1.17) enzymes transform many lipophilic compounds to more water-soluble products via conjugation with glucuronic acid. This conversion is responsible for enhancing the excretion of endogenous aglycones such as steroids. To date, several distinct isoforms of steroid UDP-glucuronosyltransferases (UGTs) have been isolated in the human liver. Among these UGTs, UGT2B7 is specific for estriol and 3,4-catechol estrogens, UGT2B15 glucuronidates 17beta-hydroxy-C19 steroids while UGT2B10 has as yet an undescribed activity. To further demonstrate the presence of UGTs in peripheral tissues we studied the expression of these enzymes in human prostate hyperplastic tissue and the LNCaP cell line. Metabolism studies using intact LNCaP cells in culture indicate the presence of UGT activities involved in the glucuronidation of 3alpha-hydroxysteroids (androsterone) and 17beta-hydroxysteroids (testosterone and dihydrotestosterone). Northern blot analysis of poly(A+) RNA from LNCaP cells and prostate using a UGT2B15 cDNA probe revealed two bands of 2.0 and 2.3 kb. In order to identify more specifically the mRNAs detected in Northern blot analysis we used RNase protection and RT-PCR, although, these approaches did not allow detection of UGT2B7 transcripts. Our studies demonstrate the presence of two UGT activities and at least two types of UGT transcripts in both the human prostate and the LNCaP.
Mol Cell Endocrinol 1995 Sep 22
PMID:Expression of transcripts encoding steroid UDP-glucuronosyltransferases in human prostate hyperplastic tissue and the LNCaP cell line. 867 24

Observation that the G protein-coupled P2U receptor (P2Y2 receptor) is activated by UTP as well as ATP provided the first indication that a class of uridine nucleotide-responsive receptors might exist. This hypothesis was confirmed by our identification of a uridine nucleotide-specific receptor on C6-2B rat glioma cells and by the recent cloning of two uridine nucleotide-responsive receptors, the P2Y6 receptor [J. Biol. Chem. 270:26152-26158 (1995)] and the P2Y4 receptor [J. Biol. Chem. 270:30849-30852 (1995) and J. Biol. Chem. 270:30845-30848 (1995)]. The relative nucleotide selectivities of these uridine nucleotide-activated receptors have not been established. Therefore, we cloned and expressed the P2Y6 and P2Y4 receptors in 1321N1 human astrocytoma cells and compared their relative selectivities for UDP, UTP, and other uridine and adenine nucleotides with that of the P2Y2 receptor expressed in the same cells. These comparisons were made by measuring inositol phosphate accumulation under conditions in which the initial purity and stability of agonists were rigidly ensured and quantitatively assessed. The data indicate that the P2Y2 receptor is activated with similar potencies by ATP and UTP but not by ADP or UDP; the P2Y6 receptor is activated most potently by UDP but weakly by UTP, ATP, and ADP; and the P2Y4 receptor is activated most potently by UTP, less potently by ATP, and not at all by nucleotide diphosphates. Furthermore, the P2Y6 receptor, which displays a uridine nucleotide selectivity essentially identical to that of the uridine nucleotide-specific receptor in C6-2B cells, was shown to be natively expressed in C6-2B cells and to account for the uridine nucleotide responses originally identified in these cells. These results define the uridine nucleotide selectivity of three phospholipase C-linked receptors: a receptor that is selectively activated by UDP (P2Y6 receptor), selectively activated by UTP (P2Y4 receptor), and activated by UTP and ATP but not by diphosphate nucleotides (P2Y2 receptor).
Mol Pharmacol 1996 Aug
PMID:Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. 870 Jan 27


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