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Query: UNIPROT:P06889 (Mol)
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S-Tubercidinylhomocysteine (STH) is a structural analog of S-adenosylhomocysteine and a potent inhibitor of S-adenosylmethionine-dependent methyltransferase reactions. We investigated the effects of STH on HeLa cell mRNA metabolism. Dual labeling studies reveal that STH dramatically inhibits the methylation of HeLa mRNA in a dose-dependent manner. Analysis of the modified nucleosides and 5'-terminal cap structures in radiolabeled mRNA by high-pressure liquid chromatography indicated that internal N6-methylation of adenosine was reduced by 65% at 50 microM STH and by 83% at 500 microM STH. The N6-methylation of adenosine contained in cap structures was similarly reduced at both concentrations of STH. Substantial amounts of cap structures lacking 2'-O-methylated nucleosides (m7GpppN, cap zero) were detected at the higher level of STH. To test the possibility that methylation affects mRNA stability, cytoplasmic mRNA half-life was measured in a pulse-chase experiment. The half-life of undermethylated mRNA, produced as a consequence of STH treatment, was unchanged compared with the control. To determine whether mRNA methylation is coupled to nuclear processing or transport, the time of cytoplasmic appearance of polyadenylated RNA in STH-treated HeLa cells was compared with untreated cells. STH caused a significant lag in the time of appearance of the polyadenylated RNA, suggesting that mRNA methylation may be required for efficient processing or transport.
Mol Cell Biol 1984 Mar
PMID:Effect of undermethylation on mRNA cytoplasmic appearance and half-life. 620 20

Transcription of cloned adenovirus, beta-globin, and retrovirus long terminal repeat DNAs in HeLa whole-cell lysate was inhibited by S-adenosylhomocysteine. However, full-length 1.7-kilobase transcripts made on adenovirus 2 late promoter DNA contained 5'-terminal GpppA, consistent with specific initiation and runoff synthesis in the absence of product methylation. Formation of runoff transcripts including retrovirus RNAs that normally contain 5'-m7GpppGmpC was not decreased by replacing GTP with non-hydrolyzable analogs, and Rous-associated virus-2 runoff products made in the presence of GTP-gamma-S contained 5'-terminal gamma-S-pppGpC. The results indicate that capping and specific transcript synthesis by RNA polymerase II are not obligatorily linked in HeLa whole-cell lysate. Accurate initiation is dependent on ATP hydrolysis, and in contrast to GTP, replacement of ATP by 5'-adenylyl-imidodiphosphate blocked specific initiation of transcripts that start with either GTP (Rous-associated virus-2, Rous-associated virus-0) or ATP (beta-globin, adenovirus).
Mol Cell Biol 1983 Dec
PMID:Initiation by RNA polymerase II and formation of runoff transcripts containing unblocked and unmethylated 5' termini. 636 23

The aliphatic adenine analogues, D-eritadenine, L-eritadenine, L-threoeritadenine, and 9-(S)-(2,3-dihydroxypropyl)adenine [(S)DHPA] function as inhibitors/inactivators of purified S-adenosylhomocysteine (AdoHcy) hydrolase, but these compounds did not induce reduction of enzyme-bound NAD+. D-Eritadenine, L-eritadenine, (S)DHPA, and L-threo-eritadenine inactivated AdoHcy hydrolase in hepatocytes, and the efficiency decreased in the order mentioned. Concurrently, there was an increase in the AdoHcy content. The accumulation of AdoHcy in the presence of (S)DHPA was more pronounced than would be expected from the inactivation of enzyme activity, suggesting that this compound may function as a reversible inhibitor as well. Furthermore, the inactivation of the intracellular enzyme by (S)DHPA is remarkable in the light of the fact that this compound induces no inactivation of purified AdoHcy hydrolase, but merely functions as an inhibitor of the enzyme. At low concentration of D-eritadenine (less than 6 microM), a distinct lag period could be demonstrated before accumulation of AdoHcy occurred. This suggests that the AdoHcy hydrolase activity must be decreased below a certain level to cause an increase in cellular AdoHcy. None of the analogues tested completely inactivated AdoHcy hydrolase and a residual enzyme activity was observed. The adenosine deaminase inhibitor, 2'-deoxycoformycin, did not potentiate the effect of these compounds on AdoHcy catabolism. The inactive enzyme formed in the presence of aliphatic adenine analogues was not reactivated under conditions where the inactivation induced by 9-beta-D-arabinofuranosyladenine was reversible.
Mol Pharmacol 1984 Nov
PMID:The effect of aliphatic adenine analogues on S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase in intact rat hepatocytes. 649 10

The 5.8S rRNA of normal tissues contains a partially 2'-O-methylated uridylic acid residue which is methylated in the cytoplasm and undermethylated in rapidly growing neoplastic tissues (R. N. Nazar, T. O. Sitz, and K. D. Somers, J. Mol. Biol., 142: 117-121, 1980). This difference in methylation was further characterized by examining the effect of cell age or cell culture passage number on the level of methylation of 5.8S RNAs from normal and malignant cell lines and simultaneous changes in intracellular pools of S-adenosylmethionine and S-adenosylhomocysteine. The results indicate that the level of methylation decreases continuously with cell culture passage number as the cells become aneuploid, transformed, or tumorigenic, but there is no direct correlation with the intracellular pools of S-adenosylmethionine or S-adenosylhomocysteine. In contrast, there is a dramatic but inverse increase in the S-adenosylmethionine:S-adenosylhomocysteine ratio which correlates with the decreasing levels of 2'-O-methylation. The significance of these changes in substrate levels to the hypomethylation of 5.8S and other RNAs during oncogenesis is discussed.
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PMID:Significance of S-adenosylmethionine pools in the hypomethylation of ribosomal RNA during the propagation of tissue culture cells and oncogenesis. 658 62

Studies on the disposition of extracellular S-adenosylhomocysteine by isolated rat hepatocytes have shown that S-adenosyl-L-homocysteine is not taken up by cells, but binds to acceptor(s) on the cell surface. The Scatchard plots for the binding of S-adenosylhomocysteine to hepatocytes and purified rat liver membranes at 0 degrees were nonlinear, and consistent with high-affinity components with Kd values of 0.4 microM and 0.7 microM, respectively. About 60% of the S-adenosylhomocysteine that was bound to cells and purified membranes dissociated rapidly from its binding sites. The rapid initial phase was followed by a second slow phase obeying first-order kinetics, corresponding to a dissociation rate constant of 0.09 min-1. S-Tubercidinylhomocysteine and unlabeled S-adenosylhomocysteine were potent inhibitors of the binding of S-[14C]adenosylhomocysteine, whereas S-3-deazaadenosylhomocysteine, S-adenosylmethionine, and S-adenosyl-D-homocysteine were less effective. A fraction of the S-adenosylhomocysteine that was bound to rat hepatocytes was displaced by low concentrations of sinefungin and its metabolite, A9145C, but these compounds were weak inhibitors of S-adenosylhomocysteine binding to purified membranes. 5'-Deoxy-5'-S-isobutylthioadenosine showed slight inhibitory activity against S-adenosylhomocysteine binding to both cells and purified membranes. In conclusion, the equilibrium binding, dissociation rate kinetics, and displacement curves in the presence of S-adenosylhomocysteine analogues show that S-adenosylhomocysteine binds to a heterogeneous population of binding sites of intact hepatocytes and purified liver plasma membranes.
Mol Pharmacol 1982 Jan
PMID:Characterization of S-adenosylhomocysteine binding to isolated rat hepatocytes and purified rat liver plasma membranes. Effect of analogues of S-adenosylhomocysteine. 713 53

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.
Mol Cell Biol 1995 Aug
PMID:Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene. 762 11

We have identified three stem abundantly expressed genes in lucerne (alfalfa, Medicago sativa). A cDNA library, constructed from lucerne stem polyadenylated RNA, was screened by differential hybridization. From this screening, cDNA clones that correspond to genes which are preferentially, or specifically, expressed in the stem were isolated. MsaS1 encodes an unidentified protein, MsaS2 encodes an S-adenosyl-homocysteine hydrolase and MsaS3 encodes an extensin-like protein. Northern blot analysis of RNA isolated from individual stem internodes indicated that the three corresponding genes show differing developmental patterns of expression. The expression of MsaS1 was confined to the youngest stem tissue and may be regulated by sucrose. In stem tissue the level of RNA for the three genes decreased in response to wounding. Tissue print hybridization analysis was used to localize the expression of the genes to the xylem side of vascular bundles in lucerne stems.
Plant Mol Biol 1995 Feb
PMID:Expression patterns of three genes in the stem of lucerne (Medicago sativa). 789 16

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy), as well as the reverse reaction, through a mechanism involving an NAD(+)-dependent oxidation of the 3'-hydroxyl group of AdoHcy (3'-oxidative activity), followed by elimination of Hcy to form 3'-keto-4',5'-didehydro-5'-deoxy-Ado. The addition of water at the 5'-position (5'-hydrolytic activity) of this tightly bound intermediate, followed by an NADH-dependent reduction, results in the formation of Ado. Based on a computer graphics model of the active site of this enzyme, it was hypothesized that amino acid residues at the carboxyl-terminal end of the protein reside in the active site of the enzyme and could play a role in catalyzing the 5'-hydrolytic reaction (Yeh, J. C., Borchardt, R. T., and Vedani, A. (1991) J. Comput. Aided Mol. Des. 5, 213-234). Using site-directed mutagenesis, we show here that lysine 426 is essential for the catalytic activity of the enzyme and that it appears to play a crucial role in the 5'-hydrolytic activity and/or stability of the quaternary structure of the human placental enzyme. Mutation of Lys-426 to arginine (K426R) produces a stable tetrameric enzyme that lacks overall catalytic activity and that was isolated predominantly as its NADH form containing tightly bound 3'-keto-Ado, suggesting that the K426R mutant has oxidative activity, but lacks 5'-hydrolytic activity, preventing it from completing the entire catalytic cycle. Mutations of Lys-426 to glutamic acid (K426E) and alanine (K426A) produce enzymes that exist primarily as monomers, do not bind NAD+ or NADH, and lack catalytic activity. The results of the Lys-426 mutations suggest that this lysine residue is crucial for the 5'-hydrolytic activity of the enzyme and/or stabilizing the quaternary structure of the enzyme.
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PMID:A single mutation at lysine 426 of human placental S-adenosylhomocysteine hydrolase inactivates the enzyme. 798 13

S-adenosylhomocysteine hydrolase (SAHase) was purified to homogeneity from the Gram negative strain Acinetobacter calcoaceticus 501. The molecular weight of the native enzyme, estimated by gel permeation, was about 288 KDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 48 KDa. The determination of the coenzyme content gave 4 mol of NAD+ and 2 mol of NADH per mol of enzyme. The isoelectric point of native SAHase was at pH 5.1. When assayed in the hydrolytic direction, the Km for S-adenosylhomocysteine and the Vmax of the enzyme for this substrate were 84 microM and 357 mumol/min/mg, respectively; in the synthetic direction, instead, the Km for adenosine and the corresponding Vmax value were 1.6 microM and 37 mumol/min/mg. Substrate analogs were tested for their ability to act as inhibitors and inactivators of the enzyme. Among these compounds, 9-beta-arabinofuranosyl adenine (Ara A) appeared as the most powerful competitive inhibitor (Ki = 18 microM) as well as the strongest time-dependent inactivator. The common feature of all the assayed analogs was the presence of the adenine ring in their molecular structure. It can thus concluded that the presence of the adenine moiety is an essential element in substrate and/or inhibitor interaction with this bacterial enzyme.
Biochem Mol Biol Int 1993 Aug
PMID:S-adenosylhomocysteine hydrolase from Acinetobacter calcoaceticus: purification and partial characterization. 822 Feb 54

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in 3H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50-1000 microM) decreased both the synthesis of labelled polyphosphoinositide (30-50%) and the release of inositol mono- and bisphosphate (40-50%). The AdoMet effect was not dependent on NE concentration (10-1000 microM), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.
Mol Cell Biochem 1993 Jul 07
PMID:S-adenosyl-l-methionine inhibits phosphoinositide metabolism in the rat brain synaptosomal suspensions. 823 76


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