Conformation of allantoicase in aqueous solution

1. The separation of 0.9-S and 10.8-S allantoicase with the aid of a 2H2O-H2O gradient was described. The resulting preparations were subjected to sedimentation equilibrium, optical rotatory dispersion (ORD), circular dichroism (CD) and infrared studies. 2. The molecular weight of 0.9-S allantoicase was determined to be about 1.1 x 10(4) g/mole in studies on the sedimentation behavior, the metal content and amino acid composition. The molecular weight of 10.8-S allantoicase was about 15.4 x 10(4) g/mole. 3. Optical rotatory dispersion, circular dichroism and infrared studies indicated that both molecules contain alpha-helix, beta conformation and random coil. A Cotton effect at 418 nm was ascribed to the asymmetric binding of Mn2+ to the enzyme. Competitive inhibitors decreased the absorption and circular dichroism bands at about 280 nm and 418 nm. These phenomena suggested that the aromatic groups may play an essential role in the binding of substrates and inhibitors by the Mn(2+)-enzyme complex. 4. Comparison of alpha-helical contents of metalloallantoicases showed that the enzymes with low helical contents exhibited high enzymic activities. 5. The nearly identical physicochemical behavior and specific enzymic activity of 0.9-S and 10.8-S allantoicase indicated that they are very similar in structure and conformation.     

Isolation and characterization of Methanoplanus endosymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus quennerstedt

Epifluorescence microscopy revealed the presence of a methanogenic bacterium as an endosymbiont in the sapropelic marine ciliate Metopus contortus. The in situ methanogenic activity of the symbiont could be demonstrated. The isolated endosymbiont was an irregular, disc-shaped bacterium with a diameter of 1.6–3.4 m. It had a generation time of 7 or 12 hours on growth on H₂/CO₂ or formate, respectively. The temperature range for growth was between 16 and 36°C with an optimum at 32°C. The optimal pH range for growth was 6.8 to 7.3. Salts, with an optimum concentration of 0.25 M, and tungsten were required for growth. The mol% G+C was 38.7%. The cell envelope consisted of proteins and a glycoprotein with an apparent molecular weight of 110,000. Morphology, antigenic relationship and the G+C content established the isolate MC1 as a new species of the genus Methanoplanus, and the name Methanoplanus endosymbiosus is proposed.Abbreviations G+C Guanine+cytosine - SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2-ethane) sulfonic acid     

Comparison of coenzyme F420 from Methanobacterium bryantii with 7- and 8-hydroxy-10-methyl-5-deazaisoalloxazine

Syntheses of 7-hydroxy-10-methyl-5-deazaisoalloxazine (7-HMDI) and 8-hydroxy-10-methyl-5-deazaisoalloxazine (8-HMDI) are described. The physicochemical and biological properties of 8-HMDI, in contrast to those of 7-HMDI, are very analogous to those of F₄₂₀, a coenzyme found in methanogenic bacteria.     

Coenzyme M derivatives and their effects on methane formation from carbon dioxide and methanol by cell extracts of Methanosarcina barkeri.

Extracts of Methanosarcina barkeri reduced methanol and CO2 to CH4 in the presence of H2 and converted methanol stoichiometrically into CH4 and CO2 in the absence of H2. In dialyzed cell-free extracts these reactions were stimulated by 2-mercaptoethanesulfonic acid (coenzyme M) and some derivatives (acetyl and formylcoenzyme M and the oxidized form of coenzyme M), which could be converted to coenzyme M by enzyme systems present in the extracts. Methylcoenzyme M could not be used in these systems.     

Association of methanogenic bacteria with rumen ciliates

In 11 species of rumen ciliates belonging to nine genera of the family Ophryoscolecidae (order Entodiniomorphida) an ectosymbiosis with methanogenic bacteria was found. The bacteria could be identified as methanogens on the basis of the presence of specific fluorescent coenzymes (F350 and F420). This somatic interaction may reflect a metabolic interaction in which efficient interspecies hydrogen transfer benefits both partners.     

Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.     

ATP synthesis from 2,3-diphosphoglycerate by cell-free extract of Methanobacterium thermoautotrophicum (strain ΔH)

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (K _i =150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme m, 2-(methylthio)ethanesulfonate - HS-HTP 7-mercaptoheptanoyl-l-threonine phosphate - CoM-SS-HTP the heterodisulfide of HS-CoM and HS-HTP - BCFE bolled cell-free extract - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - PEP phosphoenolpyruvate - 2,3-DPG 2,3-diphosphoglycerate - cDPG cyclic 2,3-diphosphoglycerate - 3-PG 3-phosphoglycerate - 2-PG 2-phosphoglycerate     

Elucidation of the structure of methanopterin, a coenzyme from Methanobacterium thermoautotrophicum, using two-dimensional nuclear-magnetic-resonance techniques

Methanopterin is a coenzyme involved in methanogenesis. From 2 kg wet cells of Methanobacterium thermoautotrophicum about 35 mumol methanopterin were isolated. The structure of this compound was elucidated by various two-dimensional nuclear-magnetic-resonance techniques. Methanopterin was identified as N-[1'-(2"-amino-4"-hydroxy-7" - methyl-6"- pteridinyl) ethyl]-4-[2',3',4',5'- tetrahydroxypent-1'- yl (5' leads to 1") O-alpha-ribofuranosyl-5"-phosphoric acid] aniline, in which the phosphate group is esterified with alpha-hydroxyglutaric acid. The molecular formula of the sodium salt of methanopterin at pH 7.0 is C30H38O16N6PNa3 X chiH2O (chi is about 4). The anhydrous sodium salt of methanopterin has a molecular mass of 838.60 Da and the molar absorption coefficient at 342 nm is 7.4 mM-1 cm-1 at pH 7.0.     

Contribution of rumen protozoa to fibre degradation and cellulase activity in vitro

Abstract The contribution of ciliates to rumen fermentation was estimated by determination of overall fibre degradation and cellulase activities (determined as carboxymethylcellulase activity) in faunated and defaunated 'artificial rumen' cultures. Experiments performed at loading rates of 22.5 and 35 g per liter per day of a grass-grain substrate revealed that fibre degradation was significantly lower in the absence of ciliates only at the high loading rate. This effect of defaunation was smaller at dilution rates below 1.7 fermenter volume turnovers per day. Bacterial numbers were higher in all experiments after removal of ciliates. Fractionation studies demonstrated that ciliates accounted for 19–28% of the total cellulase activity in faunated cultures fed on filter paper cellulose.