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PubMed1979

Properties of the methane mono-oxygenase from extracts of Methylosinus trichosporium OB3b and evidence for its similarity to the enzyme from Methylococcus capsulatus (Bath).

1. The methane mono-oxygenase from Methylosinus trichosporium OB3b was soluble. The only suitable electron donor was NAD(P)H, neither sodium L-ascorbate nor electrons derived from the oxidation of methanol could substitute for NAD(P)H. Evidence is presented for the existence of an NAD+-linked formaldehyde dehydrogenase. 2. Mono-oxygenase activity was not inhibited by a range of potential inhibitors including potassium cyanide, amytal, carbon monoxide or various metal-chelating agents, although 8-hydroxyquinoline and ethyne were effective in this respect. 3. Although the enzyme preparations were unstable on storage, the crude extract could be resolved into two components by ion-exchange chromatography. Activity could be restored to one of the components on addition of purified components from Methylococcus capsulatus (Bath). 4. Cross-reactivity of mono-oxygenase components and other similarities between the enzymes from M. trichosporium and M. capsulatus are discussed. The properties of the M. trichosporium methane mono-oxygenase reported here are contrasted with the properties of the same enzyme reported by others.

Aldehyde Oxidoreductases↗
PubMed1985

Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Aerobic stopped-flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate-limiting step for component C in the methane monooxygenase reaction. Removal and reconstitution of the redox centres of component C suggest a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH----FAD----Fe2S2----component A. This order suggests that component C functions as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for transfer to component A via the one-electron-carrying Fe2S2 centre. Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate-determining for the methane monooxygenase reaction. Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane mono-oxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.

Electrochemistry↗
PubMed1985

Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Copper(I), copper(II) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus capsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron-sulphur centre of protein C with a secondary effect at the FAD centre when the copper(II):protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron-sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule. These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.

Binding Sites↗
PubMed1989

Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath).

The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-beta-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-beta-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155-163]. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.

Detergents↗
PubMed1997

Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification.

The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-length active form and truncated forms, B' and B". Electrospray ionisation mass spectrometry (ESI-MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N-terminus of protein B. This truncate was designated B' and molecular masses were assigned to proteins B and B' of 15,852.6+/-0.4 Da and 14,629.5+/-0.3 Da, respectively. A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N-terminus of protein B. This truncate was designated B" and had a molecular mass of 12,709.93+/-0.02 Da. Proteins B' and B" were found to be inactive in the sMMO system. Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon-deficient or ompT-deficient Escherichia coli, did not inhibit B' formation. Expression of protein B as a glutathione S-transferase fusion protein and subsequent purification of protein B from E. coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild-type protein B. However, ESI-MS confirmed that protein B' was still present. Alteration of the Met12-Gly13 cleavage site to Met12-Gln13 revealed that the stability of G13Q at 20 degrees C and 37 degrees C was higher than that of wild-type preparations. ESI-MS indicated that protein B' was absent and could only be identified after prolonged incubation at room temperature. The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer. Alternatively, it may allow protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell.

Amino Acid Sequence↗
PubMed2006

Parvovirus B19 infection presenting as 'bathing trunk' erythema with pustules.

A 7-year-old girl presented with acute vulval erythema and pustules, associated with a petechial eruption in her flexures and over her feet. There was a mild prodromal illness and the patient was afebrile. There were minimal symptoms associated with the rash. Skin and throat swabs were negative and blood examination showed mild neutrophilia and lymphopaenia. Parvovirus B19 IgM was detected on serology and cutaneous features resolved within 4 days. This is a further case of parvovirus B19 infection presenting as a 'bathing trunk' exanthem that has unique dermatologic features, including the presence of pustules and distant petechiae.

Acute Disease↗
PubMed1989

Organ-bath studies using the irreversible beta-adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM).

1. The effects of the irreversible beta-adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM) were studied in isolated cardiac and uterine preparations from guinea-pigs and rats and in guinea-pig ileal preparations. 2. In the presence of BAAM (0.1-10 microM) concentration-effect curves to (-)-isoprenaline were shifted to the right in a concentration-dependent manner in all cardiac and uterine tissues. Maximum responses to (-)-isoprenaline were unaffected by BAAM except in guinea-pig left atrial and in some guinea-pig uterine preparations; however, the reductions in the maximum responses were not concentration-dependent. The mean pKB values for BAAM in guinea-pig left atria, right atria, rat whole atria and rat uterus were 7.26, 7.24, 6.84 and 7.90 respectively. 3. In guinea-pig ileal preparations, BAAM (0.1-30 microM) relaxed contractions induced by K+, histamine and acetylcholine in a non-beta-adrenoreceptor-related manner since relaxant responses were unaffected by propranolol (0.5 microM). In other tissues higher concentrations of BAAM (30-100 microM) elicited atrial standstill and depressed K+-induced contractions in uterine smooth muscle. 4. Treatment of tissues with BAAM (10-100 microM) followed by extensive wash-out increased the EC50 values for (-)-isoprenaline 21- to 83-fold. The maximum response to the catecholamine was unaffected by BAAM except in guinea-pig left atrial preparations following treatment with 100 microM BAAM. At these concentrations BAAM markedly increased the effective refractory period. 5. Concentration-effect curves for the partial agonist, oxymethylene-isoprenaline (OM-ISO) were shifted to the right 12- to 355-fold after pretreatment of tissues with BAAM (10-30 microM) followed by wash-out. The maximum response to OM-ISO was unaltered in guinea-pig and rat uteri and was reduced to a similar degree as observed with (-)-isoprenaline in guinea-pig left atria. 6. In general, the non-selective beta-adrenoreceptor antagonist BAAM depressed maximum responses to beta-adrenoreceptor agonists only in cardiac preparations and at concentrations which elicited depressant activity. On the basis of the present study, BAAM does not appear to be a suitable irreversible beta-adrenoreceptor antagonist for use in organ bath experiments.

Adrenergic beta-Antagonists↗
PubMed1989

Contractile responses to calcium chloride in rat aortic rings bathed in K+-free solution are resistant to organic calcium antagonists.

1. In rat aortic rings, devoid of functional endothelium, suspended in a modified Krebs solution (KCl: 0 mM; CaCl2: 0.63 mM), addition of CaCl2 (0.89-10 mM) produced concentration-related increases in tension (Emax = 2.38 +/- 0.10 g, EC50 = 2.31 +/- 0.15 mM, n = 36). 2. The Ca2+ evoked contractile responses were not modified by cinnarizine (10 microM), diltiazem (1 microM), ryanodine (10 microM), verapamil (1 microM), or the dihydropyridines, nitrendipine (1 microM) and (-)-Bay K 8644 (0.003-0.3 microM). 3. Cobalt chloride (0.1-1 mM) competitively antagonized the Ca2+ concentration-response curve; the Schild plot (slope 1.08 +/- 0.04), gave a pA2 value of 3.3 +/- 0.01 (n = 27). Nickel chloride (0.5-1 mM) displaced the Ca2+ concentration-response curve to the right, without an effect on the maximum response. Cadmium chloride (3-30 microM) depressed the maxima of concentration-response curves to Ca2+ with an IC50 of 15.5 +/- 1.1 microM (n = 6). 4. Monochlorobenzamil (100 microM), a Na+-Ca2+ exchange inhibitor, failed to modify the Ca2+-induced contractions. 5. In conclusion, Ca2+ evoked concentration-related contractile responses of rat aortic rings bathed in a K+-free medium; these effects were attenuated by the divalent cations cobalt, nickel and cadmium, but not modified by several organic calcium antagonists. The lack of effect of diltiazem verapamil and the dihydropyridines would suggest that, under these experimental conditions, extracellular Ca2+ enters the cytosol via pathways which are distinct from the slow (L-type) calcium channels.

Animals↗
PubMed1992

The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath).

Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

Cell Membrane↗
PubMed2001

Molecular characterization of structural genes coding for a membrane bound hydrogenase in Methylococcus capsulatus (Bath).

The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A DeltahupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo.

Bacterial Proteins↗
PubMed2005

Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath).

For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane-oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O(2). We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin (McHr) was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O(2) may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV/vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.

Amino Acid Sequence↗
PubMed2005

Identification of a copper-repressible C-type heme protein of Methylococcus capsulatus (Bath). A member of a novel group of the bacterial di-heme cytochrome c peroxidase family of proteins.

Genomic sequencing of the methanotrophic bacterium, Methylococcus capsulatus (Bath), revealed an open reading frame (MCA2590) immediately upstream of the previously described mopE gene (MCA2589). Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590-encoded protein shared significant, but restricted, sequence similarity to the bacterial di-heme cytochrome c peroxidase (BCCP) family of proteins. Two putative C-type heme-binding motifs were predicted, and confirmed by positive heme staining. Immunospecific recognition and biotinylation of whole cells combined with MS analyses confirmed expression of MCA2590 in M. capsulatus as a protein noncovalently associated with the cellular surface of the bacterium exposed to the cell exterior. Similar to MopE, expression of MCA2590 is regulated by the bioavailability of copper and is most abundant in M. capsulatus cultures grown under low copper conditions, thus indicating an important physiological role under these growth conditions. MCA2590 is distinguished from previously characterized members of the BCCP family by containing a much longer primary sequence that generates an increased distance between the two heme-binding motifs in its primary sequence. Furthermore, the surface localization of MCA2590 is in contrast to the periplasmic location of the reported BCCP members. Based on our experimental and bioinformatical analyses, we suggest that MCA2590 is a member of a novel group of bacterial di-heme cytochrome c peroxidases not previously characterized.

Amino Acid Sequence↗
PubMed1978

The effects of changes of the tonicity of the bathing fluid upon the tension generated by atrial trabeculae isolated from the heart of the frog, Rana pipiens.

Raising the tonicity of the fluid bathing frog atrial trabeculae has three effects: an initial sustained relaxation, which depends on muscle length and probably originates from structures other than the contractile apparatus; an increase in contractility, which takes the form of a transient contracture if the muscle has previously undergone a high-potassium or a low-sodium contracture, and a further rise in contractility on return to isotonic fluid (off response). The hypertonic contractures, in high-potassium or sodium-free fluids, are antagonized by local anaesthetics and in Na-free media they are unaffected by removal of extracellular coat, whereas the 'off responses' are insensitive to both experimental manoeuvres. Hypotonic fluids applied in Na-free solutions evoke a phasic and a tonic contracture, neither of which are sensitive to local anaesthetics. The tonic response is reduced by lowering the [Ca]o, and occurs at tonicities where the permeability of the cell membrane is likely to have increased. The phasic part of the hypotonic contracture resembles the 'off response' which follows exposure to hypertonic solution. The effects of hypertonic fluids and of caffeine on frog heart are alike, and are also similar to the responses induced by the same experimental manoeuvres in skeletal muscle. The results can be interpreted by assuming that the intracellular relaxing system in frog heart is sensitive to changes in tonicity, and could be functionally divided.

Animals↗
PubMed1977

Relaxation experiments using bath-applied suberyldicholine.

1. The effect of step changes in membrane potential on the end-plate conductance change produced by bath-applied suberyldicholine was studied in voltage-clamped frog muscle fibres. 2. The suberyldicholine-induced conductance increased exponentially from its previous equilibrium level to a new equilibrium level following a step hyperpolarization. 3. For low suberyldicholine concentrations the time constant of this relaxation was independent of the concentration. 4. For low suberyldicholine concentrations the voltage dependence of equilibrium conductance and relaxation time constants was identical. 5. Bungarotoxin pretreatment did not affect the responses beyond a simple reduction in their amplitude. 6. The conductance evoked by high suberyldicholine concentrations was less voltage-sensitive than that evoked by low concentrations. 7. A new model for explaining noise and relaxation data is proposed. This postulates rate-limiting binding steps followed by a voltage-dependent isomerization.

Animals↗
PubMed1978

Calcium and strontium concentration changes within skinned muscle preparations following a change in the external bathing solution.

1. A method for producing rapid [Ca2+] and [Sr2+] changes in the frog skinned muscle fibre preparation while maintaining constant all other cationic concentrations (Moisescu, 1976a, b) is described and analysed in detail. 2. Different experiments, some of them involving the Ca2+-sensitive photoprotein aequorin, as well as theoretical considerations, indicate that with this method one can produce a Ca2+ (or Sr2+) concentration change within 0.1--0.15 sec in a whole preparation having a diameter of 50 micrometer. 3. The rate of force development was similar to that observed in vivo. 4. The radial diffusion coefficient of EGTA in relaxed myofibrillar preparations was measured and found to be 4.6 x 10(-6) cm2sec-1 at 20 degrees C. 5. The sarcoplasmic reticulum in myofibrillar bundles was found to be active with respect to both Ca2+ and Sr2+ in the solutions used ([Mg2+] 1 mM; [Na] 30 mM; [K] 140-170 mM; [Cl] less than or equal to 20 mM; pH 7.10). 6. The amount of Ca released by caffeine from internal stores (previously loaded with Ca) can raise the total Ca concentration in the muscle fibre preparation by at least 1.8 mM. 7. The presence of 10 mM-caffeine in all bathing solutions reduced drastically the ability of the sarcoplasmic reticulum to accumulate both Ca and Sr.

Animals↗
PubMed2005

Quantum phase transition of a magnet in a spin bath.

The excitation spectrum of a model magnetic system, LiHoF4, was studied with the use of neutron spectroscopy as the system was tuned to its quantum critical point by an applied magnetic field. The electronic mode softening expected for a quantum phase transition was forestalled by hyperfine coupling to the nuclear spins. We found that interactions with the nuclear spin bath controlled the length scale over which the excitations could be entangled. This generic result places a limit on our ability to observe intrinsic electronic quantum criticality.

Journal Article↗
PubMed2003

The surface-associated and secreted MopE protein of Methylococcus capsulatus (Bath) responds to changes in the concentration of copper in the growth medium.

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress.

Bacterial Outer Membrane Proteins↗
PubMed1988

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from Methylococcus capsulatus bath and Methylomonas albus BG8.

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes, and in each case the protein was identified by immunoblotting with antiserum against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs.

Alcohol Oxidoreductases↗