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T C Stadtman

Publications and source records attributed to T C Stadtman.

At least 73 records · Page 4Linked to original sources
PubMed1990

In vitro incorporation of selenium into tRNAs of Salmonella typhimurium.

Broken-cell preparations of Salmonella typhimurium rapidly incorporated 75Se from 75SeO3(2-) into tRNA by an ATP-dependent process. Selenium incorporation in the presence of 50 microM 75SeO3(2-) (0.8-1 pmol per A260 unit) was enhanced by the selenocysteine precursor, O-acetyl-L-serine (to 3.7 pmol per A260 unit). This increase in incorporation was a function of O-acetyl-L-serine concentration. Neither O-acetyl-L-homoserine nor O-phospho-L-serine stimulated the incorporation of selenium into tRNA. The incorporation of 75Se from 75SeO3(2-) was decreased by adding L-selenocysteine but not by adding the D isomer. When homologous bulk tRNA was added to the broken-cell preparations, an increased rate of 75Se labeling was observed. The supernatant fraction of the broken-cell preparation contained all of the enzymes required for this process. Reversed-phase HPLC analysis of labeled bulk tRNA digested to nucleosides showed the presence of a labeled compound that coeluted with authentic 5-methylaminomethyl-2-selenouridine.

Adenosine Triphosphate↗
PubMed1990

Biosynthesis of selenium-modified tRNAs in Methanococcus vannielii.

Selenium-containing nucleosides are natural components of several tRNA species in Methanococcus vannielii. In the present study, the incorporation of selenium from 75SeO3(2-) into these macromolecules was investigated in sonic extracts of M. vannielii. Nucleoside analysis of the 75Se-labeled tRNAs from these in vitro reaction mixtures demonstrated that the selenium was present in 75Se-labeled nucleosides identical to the two naturally occurring 2-selenouridines produced in vivo. Incorporation of selenium into these nucleosides was ATP-dependent and was maximal after 20 min. Addition of O-acetylserine enhanced the activity 2- to 3-fold, implicating a role for selenocysteine in the reaction. Added L-selenocysteine could function as a selenium donor, but the D isomer and DL-selenomethionine were inactive. RPC-5 chromatography of bulk tRNA isolated from M. vannielii grown on 75SeO3(2-) separated five major species of seleno-tRNAs. The amino acid-accepting activity of these tRNAs was investigated.

Amino Acids↗
PubMed1989

Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA.

The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).

Animals↗
PubMed1989

Occurrence in vivo of selenocysteyl-tRNA(SERUCA) in Escherichia coli. Effect of sel mutations.

The selC gene of Escherichia coli codes for a novel tRNA species which is aminoacylated by L-serine and is required for the insertion of selenocysteine into proteins (Leinfelder, W., Zehelein, E., Mandrand-Berthelot, M.-A., and Böck, A. (1988) Nature 331, 723-725). As a first step toward the elucidation of the postulated pathway for selenocysteine formation from an L-serine residue esterified to tRNA, we have examined whether an increase in the selC gene dosage allows the demonstration of selenocysteyl-tRNA formation in vivo. To this end, cells of an E. coli strain carrying selC on a multicopy plasmid were labeled with [75Se]selenite, their tRNA was isolated and deacylated, and the hydrolysate was analyzed by thin layer chromatography and ion exchange chromatography. Both methods unequivocally demonstrated that the increase in the selC gene product concentration correlated with an augmented level of selenocysteine bound to tRNA. The formation of selenocysteine depended on the presence of functional products of the selA and selD genes but not of the selB gene. The selB gene product, therefore, may have a function in the decoding step itself.

Base Sequence↗
PubMed1989

Clostridial glycine reductase: protein C, the acetyl group acceptor, catalyzes the arsenate-dependent decomposition of acetyl phosphate.

The highly purified protein C component of clostridial glycine reductase is required in addition to seleno-protein A and protein B for the conversion of glycine to acetate and ammonia in the presence of arsenate. As shown by Arkowitz and Abeles [Arkowitz, R. A. & Abeles, R. H. (1989) Biochemistry 28, 4639-4644], the products are ammonia and acetyl phosphate in the presence of phosphate. The protein C component alone catalyzes an arsenate-dependent decomposition of acetyl phosphate, showing that it serves as the acetyl group acceptor in the overall reaction. A thiol-reducing agent and Mg2+ are required for catalysis of the arsenolysis reaction by protein C. Alkylation or heating at 60 degrees C completely abolishes the ability of protein C to catalyze the arsenolysis reaction and to participate as an essential component in the overall glycine reductase reaction.

Amino Acid Oxidoreductases↗
PubMed1989

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs.

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.

Aldehyde Oxidoreductases↗
PubMed1988

Selenoprotein A of the clostridial glycine reductase complex: purification and amino acid sequence of the selenocysteine-containing peptide.

A selenium-containing protein, selenoprotein A, is an essential component of the clostridial glycine reductase complex. This enzyme complex catalyzes the reductive deamination of glycine, which is coupled to the esterification of orthophosphate resulting in the formation of ATP. Sequence information was obtained by automated Edman degradation of peptides generated by digesting carboxamidomethylated selenoprotein A with chymotrypsin or trypsin or with endoproteinase Arg-C followed by Staphylococcus aureus V8 protease. The sequence near the selenocysteine (Sec) residue is -Cys-Phe-Val-Sec-Thr-Ala-Ala-Gly-Ala-Met-Asp-Leu-Glu-Asn-Glu-Lys-. Selenium-containing peptides isolated from digests of carboxamidomethylated selenoprotein A with trypsin or endoproteinase Arg-C were found to be blocked at the amino terminus. The sequence of the selenocysteine-containing peptide from selenoprotein A shows no homology with those of two other selenoproteins, glutathione peroxidase and formate dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1988

Anaerobic induction of Escherichia coli formate dehydrogenase (hydrogenase-linked) is enhanced by gyrase inactivation.

Escherichia coli synthesizes a hydrogenase-linked formate dehydrogenase (FDHH) under anaerobic conditions in the absence of nitrate. In striking contrast to many other anaerobic-specific genes, which require DNA to be negatively supercoiled for expression, we have found that inhibition of DNA gyrase activity enhances expression from the gene (fdhF) encoding the selenopolypeptide of FDHH. Fusions of the 5' flanking region of fdhF and the structural gene of lacZ were used to determine fdhF expression under varying conditions. Chemical inhibitors and a temperature-sensitive mutant allowed in vivo inhibition of gyrase activity. In each case, concomitant with gyrase inhibition there was a substantial increase in the induction of fusion protein synthesis. This enhancement of expression is observed for the intact fdhF gene residing on the chromosome as well as the fusion gene in a multicopy plasmid. Inhibition of gyrase activity will partially overcome the inhibition of fdhF expression due to nitrate but does not allow fusion protein synthesis in the presence of oxygen.

Aldehyde Oxidoreductases↗
PubMed1988

Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism.

The opal termination codon UGA is used in both prokaryotic and eukaryotic species to direct the specific insertion of selenocysteine into certain selenium-dependent enzymes. So far a formate dehydrogenase (hydrogenase-linked) of Escherichia coli and glutathione peroxidases of murine, human and rat origin have been identified as enzymes containing selenocysteine residues encoded by UGA. A novel seryl-tRNA, anticodon UCA, that specifically recognizes the UGA codon is required for selenocysteine incorporation into formate dehydrogenase. A eukaryotic UGA suppressor tRNA with UCA anticodon that accepts serine and is phosphorylated to O-phosphoseryl-tRNA may have a corresponding function in glutathione peroxidase synthesis. Other factors required for the unusual usage of the in-frame UGA codons to specify selenocysteine incorporation and the biochemical mechanism involved in distinguishing these from normal UGA termination codons are discussed.

Amino Acid Oxidoreductases↗
PubMed1988

Selenium-dependent glycine reductase: differences in physicochemical properties and biological activities of selenoprotein A components isolated from Clostridium sticklandii and Clostridium purinolyticum.

The selenoprotein A component of the glycine reductase complex of Clostridium sticklandii was shown to differ in certain properties from the selenoprotein A produced by a purine-fermenting organism, Clostridium purinolyticum. Both proteins contain one selenocysteine and two cysteine residues.

Amino Acid Oxidoreductases↗
PubMed1987

In vitro methane and methyl coenzyme M formation from acetate: evidence that acetyl-CoA is the required intermediate activated form of acetate.

Buffer-soluble extracts of acetate-grown Methanosarcina barkeri catalyzed methanogenesis from acetate in the presence of hydrogen and ATP. The rates of methane formation from either acetate plus ATP, or acetylphosphate without ATP added, were approximately doubled by the addition of coenzyme A (CoA). In vitro methyl group transfer from [2-14C]acetate to form [14CH3]methyl coenzyme M (14CH3S-CoM) was monitored by causing the accumulation of 14CH3S-CoM (14CH3-SCH2CH2SO3-) in the presence of 2-bromoethanesulfonate. The rate of 14CH3S-CoM formation was increased 2.5-fold by 0.2 mM CoA.

Acetates↗
PubMed1987

Purification and immunological studies of selenoprotein A of the clostridial glycine reductase complex.

One of the essential catalytic components of the clostridial glycine reductase complex is a selenium-containing protein, selenoprotein A. An improved method for the purification of selenoprotein A has been developed that yields milligram quantities of the protein in a few relatively simple steps. Ferredoxin and rubredoxin can be recovered in pure form as by-products of the procedure. The high resolving capabilities of an anion exchange high pressure liquid chromatography step were exploited in these purification protocols. For effective antibody production, the antigenicity of selenoprotein A was increased by coupling the pure protein via its sulfhydryl/selenol group(s) to the amino groups of bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester. The high titer sheep antisera that were elicited were used to study the mechanisms of selenium incorporation into selenoprotein A. Immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels was employed to monitor the synthesis of selenoprotein A by Clostridium sticklandii as a function of growth conditions. Cells grown under limiting conditions (1 nM) of selenium contained only 1-2% of normal levels of active selenoprotein A and no precursor forms were detected after DEAE-high pressure liquid chromatography fractionation of extracts. Conversely, cells grown with optimal (1 microM) selenium levels contained maximal amounts of seleno-protein A and expressed full glycine reductase activity.

Amino Acid Oxidoreductases↗
PubMed1987

Carbon monoxide dehydrogenase from Methanosarcina barkeri. Disaggregation, purification, and physicochemical properties of the enzyme.

Carbon monoxide dehydrogenase from acetate-grown cells of Methanosarcina barkeri exists in a high molecular weight form (approximately 3 X 10(6)) under conditions of high ionic strength but is converted to a much smaller form by dialysis. The enzyme was purified by a procedure which exploits isolation of the aggregated form by gel filtration and subsequent dissociation. Following this, the enzyme was purified to within 92% of homogeneity by chromatography on phenyl-Sepharose and finally on hydroxylapatite. Due to the extreme oxygen lability of the enzyme, the entire procedure was carried out within the anaerobic laboratory at the National Institutes of Health. The enzyme has an alpha 2 beta 2 oligomeric structure composed of subunits with molecular weights of 19,700 and 84,500. The amino acid compositions of the individual subunits were determined. Analysis of the metal content by plasma emission spectroscopy indicated 1.3 +/- 0.3 (n = 4) nickel and 15.6 +/- 5.6 (n = 5) iron per mol of alpha 2 beta 2. The enzyme did not contain significant amounts of cobalt or molybdenum. Ferredoxin, FAD, FMN, 2,3,5-triphenyltetrazolium chloride, methyl viologen, and phenazine methosulfate served as electron acceptors; however, the enzyme failed to reduce NAD+, NADP+, or the 8-hydroxy-5-deazaflavin factor F420. The optimum pH was between 7 and 9. The apparent Km for methyl viologen was 7.1 mM, whereas the value for 2,3,5-triphenyltetrazolium chloride was below 0.5 mM. Strong inhibition was observed by oxygen and cyanide. Inactivation by glyoxaldehyde required enzymatic turnover which suggested that a reactive group was formed, or exposed, on an enzyme intermediate in catalysis. A high degree of thermostability was noted. Carbon monoxide, however, rendered the enzyme more susceptible to temperature inactivation.

Aldehyde Oxidoreductases↗
PubMed1987

Purification of individual tRNAs using a monoclonal anti-AMP antibody affinity column.

A murine monoclonal anti-AMP antibody affinity matrix was used for isolation of individual species of amino acid transfer nucleic acids (tRNAs). The antibodies had been prepared using 5'-AMP covalently attached to bovine serum albumin as antigen and exhibited high affinity for 5'-AMP but greatly reduced affinity for 3'-AMP. Native uncharged tRNAs that terminate in a 5'-AMP group on the amino acid acceptor arm of the molecule bind tightly to the anti-AMP affinity matrix, whereas aminoacylated tRNAs are not retained. This allows separation of a particular tRNA species as its aminoacyl derivative from a complex mixture of uncharged tRNAs under very mild conditions.

Adenosine Monophosphate↗
PubMed1987

Solubilization of a membrane-bound diol dehydratase with retention of EPR g = 2.02 signal by using 2-(N-cyclohexylamino)ethanesulfonic acid buffer.

A procedure for solubilization of the oxygen-labile, membrane-bound diol dehydratase from Clostridium glycolicum with retention of enzymatic activity is described. The procedure involves sonication of crude membrane preparations anaerobically in 0.1 M 2-(N-cyclohexylamino)ethane-sulfonic acid (CHES) buffer (pH 8.6-9.0) containing 2 mM dithiothreitol. The addition of dimethylsulfoxide (30%) and lysophosphatidylcholine (0.15 mg/ml) to the solubilization buffer resulted in a 10-fold increase in recovery of solubilized diol dehydratase activity. After ultracentrifugation, an overall recovery of 50% of the activity initially present in the crude membrane preparations was achieved. Active membrane preparations and the solubilized enzyme exhibited an EPR signal at g = 2.02. Both enzyme activity and EPR signal were sensitive to oxygen and the radical scavengers, NH2OH and hydroxy-urea.

Buffers↗
PubMed1987

Specific occurrence of selenium in enzymes and amino acid tRNAs.

In contrast to the widespread ability of bacteria, plants, and animals to incorporate selenium nonspecifically into proteins in the form of selenomethionine residues, the selenoamino acid selenocysteine occurs as a highly specific component of a few selenium-dependent enzymes. Selenocysteine has been identified in glycine reductase, formate dehydrogenase, and hydrogenase of bacterial origin and glutathione peroxidase from mammalian and avian sources. In these enzymes there is evidence that the selenol group, which is largely ionized at physiological pH, functions as a redox center. It now seems clear, from studies with both prokaryotes and eukaryotes, that the UGA opal stop codon is used to specify the cotranslational insertion of selenocysteine into proteins. The factors that allow this unusual use of the stop codon are, however, unknown. The occurrence of selenium as a normal constituent of several bacterial tRNA species has been established. The presence of a selenonucleoside, 5-methylaminomethyl-2-selenouridine, in the first or wobble position of the anticodons of certain glutamate and lysine iso-acceptor species influences codon-anticodon interaction and thus may serve to regulate translational processes. The biosynthesis of the selenonucleoside appears to involve the ATP-dependent activation of the sulfur in a preformed 5-methylaminomethyl-2-thiouridine residue in tRNA and replacement of the sulfur with selenium.

Animals↗