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T C Stadtman

Publications and source records attributed to T C Stadtman.

At least 109 records · Page 6Linked to original sources
PubMed1980

Reconstitution of a formate-NADP+ oxidoreductase from formate dehydrogenase and a 5-deazaflavin-linked NADP+ reductase isolated from Methanococcus vannielii.

The formate-dependent reduction of NADP+ by extracts of Methanococcus vannielii is catalyzed by a coupled system consisting of formate dehydrogenase, a 5-deazaflavin cofactor, and 5-deazaflavin-dependent NADP+ reductase. All three components were purified from crude extracts of M. vannielii. Recombination of these components reconstituted the formate-NADP+ oxidoreductase system. The formate dehydrogenase also can utilize FAD, FMN, and a number of artificial dyes as electron acceptors, but these do not replace the 5-deazaflavin cofactor in the coupled enzyme system. The reduced form of 5-deazaflavin binds readily to the NADP+ reductase apoprotein and is not dissociated by ammonium sulfate treatment at neutral pH under anaerobic conditions. This electron transfer cofactor from M. vannielii is identical in many of its properties to the 5-deazaflavin isolated from other methane-producing bacteria.

Aldehyde Oxidoreductases↗
PubMed1980

Selenium-containing tRNAs from Clostridium sticklandii: cochromatography of one species with L-prolyl-tRNA.

75Se-Labeled tRNAs were synthesized by Clostridium sticklandii cultures supplemented with 1 microM sodium [75Se]selenite or [75Se]selenocysteine. This process is highly specific for selenium; it occurred in the presence of 1.2 mM sodium sulfide and was not decreased by the further addition of a 500-fold molar excess of cysteine. The 75Se in these tRNAs was located in the polynucleotide portion of the molecules and not in esterified (alkali-labile) selenocysteine. Inhibition of cell multiplication by antibiotics that block either protein synthesis or DNA-dependent RNA synthesis did not prevent this 75Se incorporation. Three [75Se]tRNAs were separated from C. sticklandii cells labeled in the presence of chloramphenicol and were partially purified by chromatography on benzoylated DEAE-cellulose and DEAE-Sephadex A-50 columns. These were designated seleno-tRNAs I, II, and III according to their elution sequence from benzoylated DEAE-cellulose. Cochromatography of purified seleno-tRNA II on DEAE-Sephadex A-50 with an L-proline-accepting species suggests that it is a selenium-containing L-prolyl-tRNA.

Chromatography↗
PubMed1980

Selenium-dependent enzymes.

Selenium, molecular weight 78.96, resembles sulfur in many of its chemical properties and occurs in inorganic forms as H2Se, H2Se2O3, H2SeO3, and H2SeO4 which are the analogues of hydrogen sulfide, thiosulfate, sulfite, and sulfate, respectively. The commonly available radionuclide, 75Se, is a gamma emitter (half-life 122 days) that is used extensively as a tracer in biochemical studies and as a radiopharmaceutical agent for diagnostic purposes. Organoselenium compounds, in general, are less stable and more reactive than the corresponding sulfur analogues and these properties may account for the toxicity of selenium when it is incorporated indiscriminately in place of sulfur in cellular constituents. On the other hand living systems may have exploited the greater reactivity of certain types of organoselenium compounds in those instances where selenium is specifically required as a component of an enzyme or other macromolecule. Several enzymic processes that do not distinguish selenium from sulfur and therefore may be important in selenium toxicity were discussed in some detail in two earlier reviews on selenium biochemistry (1, 2) and this aspect of the problem is not treated here. Rather, the information currently available on the properties and catalytic functions of the four known selenium-dependent enzymes is summarized. These enzymes are formate dehydrogenases of Escherichia coli and several anaerobic bacteria, clostridial glycine reductase, mammalian and avian glutathione peroxidase, and nicotinic acid hydroxylase of Clostridium barkeri. Additional selenoproteins whose catalytic activities are as yet unidentified are mentioned.

Aldehyde Oxidoreductases↗
PubMed1977

Methanococcus vannielii: ultrastructure and sensitivity to detergents and antibiotics.

Methanococcus vannielii is a strictly anaerobic motile coccus that possesses a tuft of flagellae. The cells are markedly sensitive to mechanical stress and are readily lysed by detergents, but the organism grows normally in media of low ionic strength. The absence of a typical cell wall, further suggested by resistance of M. vannielii to penicillin, cycloserine, and vancomycin, was confirmed by ultrastructural studies. Electron micrographs showed that the cell envelope lacks a peptidoglycan layer. On the outer surface there is a regular array of subunits similar to those of the glycoprotein envelopes of the halobacteria. However, the M. vannielii cell envelope, unlike those of the holobacteria, is unable to maintain a definite shape, and a high salt concentration is not required for its integrity.

Anti-Bacterial Agents↗
PubMed1976

Purification and properties of proline reductase from Clostridium sticklandii.

Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.

Amino Acid Oxidoreductases↗
PubMed1976

Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety.

A small, heat-stable selenoprotein, one of the components of the glycine reductase complex, was labeled with 75Se by growth of Clostridium sticklandii in the presence of Na2 75SeO3. The selenium-containing moiety, which is essential for the biological activity of the protein, was shown to be a selenocysteine residue. It was isolated as its Se-carboxymethyl, Se-carboxyethyl, and Se-aminoethyl derivatives from digests of the pure 75Se-labeled protein that had been reduced and treated with the various alkylating agents prior to hydrolysis. In each instance the 75Se-labeled moiety obtained from an alkylated protein sample and the corresponding alkyl derivative of authentic selenocysteine were indistinguishable. Several studies of the native selenoprotein detected a chromophore (UVmax 238nm) that appeared upon reduction of the protein with KBH4 and rapidly disappeared upon exposure to oxygen. This oxygen-labile chromophore is thought to be the ionized -SeH group of the selenocysteine residue.

Alkylating Agents↗
PubMed1974

Selenium biochemistry.

The toxicity of selenium to animals and plants has been known and extensively documented since the 1930's, but it is only during the past 15 years that selenium has also been shown to be an essential micronutrient for animals and bacteria. Very little is known about the specific role or roles of selenium and, to date, there are only three enzyme-catalyzed reactions that have been shown to require the participation of a selenium-containing protein. These are the reactions catalyzed by (i) formate dehydrogenase of bacteria, (ii) glycine reductase of clostridia, and (iii) glutathione peroxidase of erythrocytes. The common denominator of these selenium-dependent processes is that they are all oxidation-reduction reactions. A fourth selenoprotein has been isolated from skeletal muscle of sheep but its catalytic function has not been identified. The form in which selenium occurs in these selenoproteins is unknown. The selenoprotein of clostridial glycine reductase contains selenium in a covalently bound form. Studies in progress indicate that this may be an organoselenium compound not previously detected in nature. Identification of the chemical nature of selenium in proteins participating in electron transport processes should enable us to determine its specific role and to understand the basic defects in certain cardiac and skeletal muscle degenerative diseases which are selenium-deficiency syndromes. The greater availability and ease of isolation of the selenoprotein of the bacterial glycine reductase system makes this the biological material of choice for studies on the mechanism of action of selenium. An added attractive feature of this system is that it can conserve the energy made available by the reductive deamination of glycine in a biologically useful form by synthesizing ATP.

Adenosine Triphosphate↗