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Biomedical subjects

R S Wolfe

Publications and source records attributed to R S Wolfe.

At least 91 records · Page 5Linked to original sources
PubMed1980

Methyl coenzyme M reductase from Methanobacterium thermoautotrophicum. Resolution and properties of the components.

Oxygen-labile extract of Methanobacterium thermoautotrophicum was resolved into three components, A, B, and C, that were required for the reductive demethylation of methyl coenzyme M, 2-(methylthio)-ethanesulfonate, in the presence of molecular hydrogen. Components A and C were found to be large heat-labile proteins with A being in excess of 500,000 daltons, and C being 130,000 daltons. Component A exhibited hydrogenase activity for reduction of viologen dyes, coenzyme F420, or flavins but not for NAD+ or NADP+. An apparent Km of 25 microM was determined for F420 and a Km of 1.5 mM for methyl viologen. After centrifugation at 100,000 x g for 1 h, 80% of Component A was found in the supernatant solution. Component B was found to be an oxygen-labile, heat-resistant, dialyzable cofactor with a size of about 1,000 daltons and with no apparent absorption in the visible range. Known cofactors failed to substitute for the new coenzyme.

Euryarchaeota↗
PubMed1980

Methane formation from fructose by syntrophic associations of Acetobacterium woodii and different strains of methanogens.

When Acetobacterium woodii was co-cultured in continuous or in stationary culture with Methanobacterium strain AZ, fructose instead of being converted to 3 mol of acetate was converted to 2 mol of acetate and 1 mol each of carbon dioxide and methane, showing that interspecies hydrogen transfer occurred. In continuous culture the organisms formed a close physical association in clumps; the doubling time for each organism was 6 h at 33 degrees C. Methane mainly was derived from carbon positions 3 and 4 of the sugar, but other carbons also yielded methane; this was shown to be due to carbon dioxide-acetate exchange reactions by A. woodii in a manner similar to that carried out by Clostridium thermoaceticum. Four other methanogens, Methanobacterium M.o.H. and M.o.H. G, Methanobacterium formicicum, and Methanosarcina barkeri (not acetate-adapted) also produced similar results, when co-cultured with A. woodii.

Acetobacter↗
PubMed1980

Nitrate dissimilation under microaerophilic conditions by a magnetic spirillum.

During microaerophilic growth of magnetic spirillum MS-1 on tartrate and nitrate, a maximal cell density was obtained at an initial oxygen partial pressure of 17 Pa. A transient accumulation of nitrous oxide and a 1:2 (mol/mol) stoichiometry between tartrate oxidation and nitrate reduction were observed, indicating that the organism carried out a respiratory type of metabolism.

Journal Article↗
PubMed1980

Methyl-coenzyme M, an intermediate in methanogenic dissimilation of C1 compounds by Methanosarcina barkeri.

Extracts of Methanosarcina barkeri possess a specific methyltransferase that catalyzes the transfer of the methyl group of methanol to 2-mercaptoethanesulfonic acid. Over a fourfold range in added 2-mercaptoethanesulfonic acid, the formation of 2-(methylthio)ethanesulfonic acid exhibited a 1:1 ratio to 2-mercaptoethanesulfonic acid added. This reaction required adenosine 5'-triphosphate; a maximal ratio (mole/mole) of 85 methyl groups was transferred per adenosine 5'-triphosphate added. The methyltransferase was found in extracts of methanol-grown cells as well as in extracts of hydrogen-grown cells. In extracts of cells grown on either substrate, 2-(methylthio)ethanesulfonic acid was formed from added methanol or methylamine but not from acetate.

Adenosine Triphosphatases↗
PubMed1979

Complete degradation of carbohydrate to carbon dioxide and methane by syntrophic cultures of Acetobacterium woodii and Methanosarcina barkeri.

Methanosarcina barkeri (strain MS) grew and converted acetate to CO2 and methane after an adaption period of 20 days. Growth and metabolism were rapid with gas production being comparable to that of cells grown on H2 and CO2. After an intermediary growth cycle under a H2 and CO2 atmosphere acetate-adapted cells were capable of growth on acetate with formation of methane and CO2. When acetate-adapted Methanosarcina barkeri was co-cultured with Acetobacterium woodii on fructose or glucose as substrate, a complete conversion of the carbohydrate to gases (CO2 and CH4) was observed.

Acetates↗
PubMed1979

Specificity and biological distribution of coenzyme M (2-mercaptoethanesulfonic acid).

The specificity of the growth requirement of Methanobacterium ruminantium strain M1 for a new coenzyme, 2-mercaptoethanesulfonic acid (HS--CoM), was examined. A variety of derivatives, analogs, and potential biosynthetic precursors of coenzyme M were tested; only a restricted range of thioether, thioester, and thiocarbonate derivatives of the cofactor were found to replace the HS--CoM requirement. Bromoethanesulfonic acid (BrCH2CH2SO3-), a halogenated analog of HS--CoM, potently inhibited the growth response. No coenzyme was detectable in a wide range of nonmethanogenic eucaryotic tissues and procaryotic organisms. However, all methanogens available in pure culture exhibited high levels of coenzyme M which ranged from 0.3 to 16 nmol/mg of dry weight.

Bacteria↗
PubMed1979

Transport of coenzyme M (2-mercaptoethanesulfonic acid) in Methanobacterium ruminantium.

A system for transport of coenzyme M, 2-mercaptoethanesulfonic acid (HS--CoM), in Methanobacterium ruminatium strain M1 required energy, showed saturation kinetics, and concentrated the coenzyme against a gradient. The process was sensitive to temperature and was maximally active at pH 7.1. Cells took up HS--CoM at a linear rate, with a Vmax of 312 pmol/min per mg (dry weight) and an apparent Km of 73 nM. An intracellular pool of up to 5 mM accumulated which was not exchangeable with the medium. Uptake required both hydrogen and carbon dioxide; it was inhibited by O2. Bromoethanesulfonic acid (BrCH2CH2SO3-), a potent inhibitor of methanogenesis in cell-free extracts, inhibited both uptake and methane production. Results of inhibitor studies with derivatives and analogs of the coenzyme showed that the specificity of the carrier is restricted to a limited range of thioether, thioester, and thiocarbonate derivatives. 2-(Methylthio)ethanesulfonic acid (CH3--S--CoM) showed an apparent Ki for HS--CoM uptake of 15 nM, being taken up itself with a Vmax of 320 pmol/min per mg (dry weight) and an apparent Km of 50 nM. An analysis of intracellular pools after HS--CoM uptake indicated that the predominant forms are a heterodisulfide of unknown composition and CH3--S--CoM.

Biological Transport, Active↗
PubMed1979

Distribution of coenzyme F420 and properties of its hydrolytic fragments.

The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system.

Acetobacter↗
PubMed1979

Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium.

A bipolarly flagellated magnetotactic spirillum containing intracellular chains of single domain-sized magnetite crystals was isolated by applying a magnetic field to sediments from a freshwater swamp. The organism was cultured in a chemically defined medium containing ferric quinate and succinate as sources of iron and carbon, respectively. Nonmagnetic variants of this isolate were maintained in chemically defined medium lacking ferric quinate. In contrast to magnetic cells, these had less iron and lacked measurable magnetic remanence and the intracytoplasmic crystals. In other respects, including moles percent guanine plus cytosine content, growth characteristics, nutrition, and physiology, the two types were similar. The isolate reduced nitrate without accumulating nitrite and produced ammonia during growth. Nitrate or ammonium ions served as a nitrogen source. The organism was microaerophilic and did not grow anaerobically with nitrate in the medium. In chemically defined medium, cells synthesized magnetite only if the initial O2 concentration in the atmosphere of sealed cultures was 6% (vol/vol) or less.

Crystallization↗
PubMed1978

Proposed structure for coenzyme F420 from Methanobacterium.

The low-potential electron carrier, coenzyme F420, was purified from Methanobacterium strain M.o.H. A yield of 160 mg/kg of wet-packed cells was obtained. Results of analysis of hydrolytic fragments and periodate oxidation products of the coenzyme, by infrared, UV-visible, 1H and 13CNMR spectrometry, mass spectrometry, and quantitative elemental analyses indicate that coenzyme F420 is: N-[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy-4-pentoxyhydroxyphosphinyl]-L-lactyl]-gamma-L-glutamyl]-L-glutamic acid. A convenient trivial name would be the N-(N-L-lactyl-gamma-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate. Proof of structure by organic synthesis was not performed; the stereochemical configuration of the hydroxyl groups on the side chain as well as the position of the hydroxyl group on the aromatic ring require confirmation by organic synthesis of the molecule.

Alkaline Phosphatase↗
PubMed1978

Preparation of coenzyme M analogues and their activity in the methyl coenzyme M reductase system of Methanobacterium thermoautotrophicum.

A number of 2-(methylthio)ethanesulfonate (methyl-coenzyme M) analogues were synthesized and investigated as substrates for methyl-coenzyme M reductase, an enzyme system found in extracts of Methanobacterterium thermoautotrophicum. Replacement of the methyl moiety by an ethyl group yielded an analogue which served as a precursor for ethane formation. Propyl-coenzyme M, however, was not converted to propane. Analogues which contained additional methylene carbons such as 3-(methylthio)propanesulfonate or 4-(methylthio)butanesulfonate or analogues modified at the sulfide or sulfonate position, N-methyltaurine and 2-(methylthio)ethanol, were inactive. These analogues, in addition to a number of commercially available compounds, also were tested for their ability to inhibit the reduction of methyl-coenzyme M to methane. Bromoethanesulfonate and chloroethanesulfonate proved to be potent inhibitors of the reductase, resulting in 50% inhibition at 7.9 X 10(6) M and 7.5 X 10(5) M. Analogues to coenzyme M which contained modifications to other regions were evaluated also and found to be weak inhibitors of methane biosynthesis.

Bacteria↗