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1.
The separation of two distinct forms of cytochrome P450 from adrenal cortex mitochondria has been achieved by the following steps; (1) lyophilisation (2) iso-octane extraction, (3) (NH(4))(2)SO(4) fractionation in the presence of sodium cholate. The fraction precipitating between 25-35 percent (NH(4))(2)SO(4) gave a difference spectrum with 11-deoxycorticosterone (11-DOC) but not with 20alpha-hydroxycholesterol (20alpha-HOC). This fraction showed high 11beta-hydroxylase activity but low activity for side chain cleavage of cholesterol (S.C.C.). The fraction precipitating between 45-60 percent (NH(4))(2)SO(4) gave a difference spectrum with 20alpha-HOC but not with 11-DOC and exhibited high S.C.C. activity but low 11beta-hydroxylase activity. The absorption spectrum of the 45-60 percent fraction indicated a preponderance of high spin hemoprotein (lambda(max) 395 nm). 相似文献
2.
Physiological Studies of Methane and Methanol-Oxidizing Bacteria: Oxidation of C-1 Compounds by Methylococcus capsulatus 总被引:18,自引:12,他引:6
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Methylococcus capsulatus grows only on methane or methanol as its sole source of carbon and energy. Some amino acids serve as nitrogen sources and are converted to keto acids which accumulate in the culture medium. Cell suspensions oxidize methane, methanol, formaldehyde, and formate to carbon dioxide. Other primary alcohols are oxidized only to the corresponding aldehydes. Oxidation of formate by cell suspensions is more sensitive to inhibition by cyanide than is the oxidation of other one carbon compounds. This is due to the cyanide sensitivity of a soluble nicotinamide adenine dinucleotide-specific formate dehydrogenase. Oxidation of formaldehyde and methanol is catalyzed by a nonspecific primary alcohol dehydrogenase which is activated by ammonium ions and is independent of pyridine nucleotides. Some comparisons are made with a strain of Pseudomonas methanica. 相似文献
3.
S ummary : Spontaneous mutants of Methylococcus capsulatus resistant to antibiotics, amino acid analogues and other compounds were obtained at frequencies similar to those found in other bacteria. Attempts to increase these frequencies with the mutagens N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroso-N-methyl urethane, ethyl methane-sulphonate and UV were unsuccessful. Using these mutagens, only one auxotrophic mutant was isolated from 11,082 colonies examined. The growth characteristics of this p -aminobenzoic acid requiring mutant are described. 相似文献
4.
Helle N. Carlsen Lars Joergensen Hans Degn 《Applied microbiology and biotechnology》1991,35(1):124-127
Summary The kinetics of methane uptake by Methylococcus capsulatus (Bath) and its inhibition by ammonia were studied by stopped-flow membrane-inlet mass spectrometry. Measurements were done on suspensions of cells grown in high- and low-copper media. With both types of cells the kinetics of methane uptake are hyperbolic when oxygen is in excess. The apparent K
m and K
max for methane uptake are both higher in low-copper cells than in high-copper cells. Ammonia is a simple competitive inhibitor of methane uptake in high-copper cells when the oxygen concentration is above a few M. The findings agree with the assumption that ammonia is a week alternative substrate for particulate methane monooxygenase. In low-copper cells the effect of ammonia is complicated and cannot be explained in terms of current assumptions on the mechanism of soluble methane monooxygenase. Our data indicate that ammonia inhibition is likely to be a more serious problem in connection with cultivation in low-copper medium than in high-copper medium.
Offprint requests to: H. N. Carlsen 相似文献
5.
Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath. 总被引:2,自引:1,他引:1
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An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline. 相似文献
6.
Inhibition of dimethyl ether and methane oxidation in Methylococcus capsulatus and Methylosinus trichosporium. 总被引:2,自引:2,他引:0
Metal-chelating or -binding agents inhibited the oxidation of dimethyl ether and methane, but not methanol, by cell suspensions of Methylococcus capsulatus and Methylosinus trichosporium. Evidence suggests that the involvement of metal-containing enzymatic systems in the initial step of oxidation of dimethyl ether and methane. 相似文献
7.
The fine structure of Methylococcus capsulatus is described. Particular emphasis is focused on the intracytoplasmic membrane system which is organized as a stacked array of flattened saccules. Each saccule is limited by a 75 A unit membrane and lies in close apposition to adjacent saccules. Methylococcus capsulatus is an obligate methylotroph whose sole source of carbon and energy is methane (or methanol). In this study methane oxidation is demonstrated for the first time in a cell-free system. Work is in progress to determine the cellular organelles which constitute the particulate fraction responsible for methane oxidation. The possible role of the intracytoplasmic membranes in energy transfer is considered in relation to the functions of stacked membrane arrays in other animal, plant and bacterial systems. 相似文献
8.
S ummary : Experiments were directed towards the production of high biomass concentrations in cultures of Methylococcus capsulatus. In shake flasks the effects of ammonium ion, phosphate ion and various trace metals on growth were studied. In the chemostat the effects on growth of methane limitation, oxygen limitation, aeration, dilution rate, pH value and temperature were studied and carbon balances were made in steady state conditions. Growth in the fermenter was stimulated by the use of Amberlite CG–120 ion exchange resin in the medium. The probability that Amberlite removed a growth, inhibitor is discussed. 相似文献
9.
Synthesis of cell constituents by methane-grown Methylococcus capsulatus and Methanomonas methanooxidans 总被引:19,自引:8,他引:11
1. A study was made of the incorporation of carbon from [(14)C]methanol by cultures of Methylococcus capsulatus and Methanomonas methanooxidans growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled substrate for periods of up to 3min, was analysed by chromatography and radioautography. 3. Over 80% of the radioactivity fixed by Methylococcus capsulatus at 30 degrees C at the earliest times of sampling appeared in phosphorylated compounds, of which glucose phosphate constituted 60%. 4. Most of the radioactivity fixed by Methanomonas methanooxidans at 30 degrees C at the earliest times of sampling appeared in serine, malate, aspartate and an unknown compound(s) tentatively suggested to be folate derivative(s). At 16 degrees C, [(14)C]methanol was fixed predominantly into serine and the unknown compound(s). 5. Extracts of Methylococcus capsulatus contain an enzyme system that catalyses the condensation of formaldehyde and ribose 5-phosphate to give a mixture consisting mainly of fructose phosphate and allulose phosphate. No similar activity was detected in extracts of Methanomonas methanooxidans. A convenient method was developed for assay of this enzyme system. 6. The enzyme system catalysing the condensation of formaldehyde with ribose 5-phosphate is particle-bound in both Methylococcus capsulatus and Pseudomonas methanica and is unstable in the absence of Mg(2+). 7. Extracts of Methanomonas methanooxidans contain high activities of d-glycerate-NAD oxidoreductase, whereas extracts of Methylococcus capsulatus and Pseudomonas methanica contain negligible activities of this enzyme. 8. These results indicate that during growth of Methylococcus capsulatus on methane, as with Pseudomonas methanica, cell constituents are made by the ribose phosphate cycle of formaldehyde fixation. This contrasts with Methanomonas methanooxidans, whose assimilation pathway resembles in some features that of Pseudomonas AM1 growing on methanol. 相似文献
10.
11.
A method for reducing RNA and DNA in the bacterium Methylococcus capsulatus (Bath) has been developed. Endogenous RNase and DNase were activated by a 10 s heat shock at 90°C. Cells were then incubated
at 60°C for 20 min to allow degradation of the nucleic acids. The optimum pH for the process was 7.0. The protein loss was
less than 10% and occurred during the initial heat shock. No protein loss was found during incubation. The total dry-weight
loss in connection with an 80% reduction of the nucleic acid content was 20%–25%, giving a final product with a raw protein
content of approximately 75%. Reduction of both RNA and DNA was inhibited by CuSO4 and ZnSO4. DNA reduction was stimulated by other minerals. Optimal stimulation was found at 1 mM FeSO4. Reduction of RNA was not increased by any of the minerals tested.
Received: 29 June 1995/Received last revision: 2 October 1995/Accepted: 16 October 1995 相似文献
12.
Valentine AM LeTadic-Biadatti MH Toy PH Newcomb M Lippard SJ 《The Journal of biological chemistry》1999,274(16):10771-10776
Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling. 相似文献
13.
Anaerobic methane oxidation is a general process important in controlling fluxes of methane from anoxic marine sediments. The responsible organism has not been isolated, and little is known about the electron acceptors and substrates involved in the process. Laboratory evidence indicates that sulfate reducers and methanogens are able to oxidize small quantities of methane. Field evidence suggests anaerobic methane oxidation may be linked to sulfate reduction. Experiments with specific inhibitors for sulfate reduction (molybdate), methanogenesis (2-bromoethanesulfonic acid), and acetate utilization (fluoroacetate) were performed on marine sediments from the zone of methane oxidation to determine whether sulfate-reducing bacteria or methanogenic bacteria are responsible for methane oxidation. The inhibition experiment results suggest that methane oxidation in anoxic marine sediments is not directly mediated by sulfate-reducing bacteria or methanogenic bacteria. Our results are consistent with two possibilities: anaerobic methane oxidation may be mediated by an unknown organism or a consortium involving an unknown methane oxidizer and sulfate-reducing bacteria. 相似文献
14.
Oxidation of C1 compounds by particulate fractions from Methylococcus capsulatus: properties of methanol oxidase and methanol dehydrogenase. 总被引:12,自引:6,他引:6
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Methanol (and formaldehyde) oxidizing activities in crude extracts of Methylococcus capsulatus are associated mainly with particulate fractions sedimenting between 3,000 and 40,000 X g. Most of the phenazine methosulfate (PMS)-dependent methanol (and formaldehyde) dehydrogenase activity observed resides in the soluble fraction but represents only 40% of the total (PMS dependent plus independent) activity. Both PMS-dependent methanol dehydrogenase activity and PMS-independent methanol oxidase activity are found in particulate fractions, and the PMS-dependent dehydrogenase is easily solubilized by treatment with certain phospholipases or detergents. The properties of the PMS-dependent dehydrogenase activities in the soluble fraction and that solubilized from the particles suggested that they may be identical proteins. Their pH optima, temperature dependence, thermolabilities, and sensitivities to the presence of specific antisera were indistinguishable. Homogeneous preparations of the enzyme proteins obtained from the soluble fractions of extracts and the particulate fractions solubilized by detergents had similar: (i) electrophoretic mobilities in native and denatured states (subunit size in sodium dodecyl sulfate 62,000 daltons); (ii) molecular radii under native conditions, (iii) visible absorption spectra, lambdamax 350 nm, (iv) kinetic constants for methanol and formaldehyde; (v) substrate specificity; and (vi) immunological characteristics--antisera to each enzyme preparation showed precipitin lines of identity to either of the enzymes. It is suggested that the major site of methanol and formaldehyde oxidation in M. capsulatus occurs on the intracytoplasmic membranes in vivo and is coupled to oxygen reduction. 相似文献
15.
M B Kemp 《The Biochemical journal》1972,127(3):64P-65P
16.
A. G. Zhivotchenko E. S. Nikonova M. H. Jørgensen 《Bioprocess and biosystems engineering》1995,14(1):9-15
Nitrogen fixation has been investigated during chemostat fermentations with a culture of Methylococcus capsulatus with natural gas. It is demonstrated that nitrogen fixation occurs under conditions when either nitrate or ammonia as nitrogen source is insufficient for the growth on fixed supply of methane and oxygen. The fixation occurs contrary to expectations within a wide range of dilution rates and with variation of concentration of liquid source of nitrogen. An O2 optimum is determined for the nitrogenase system of the culture in an assay. During fermentation a complete abolishment of nitrogenase reaction is attained at 15% air saturation (dissolved oxygen). Conditions for N2 fixation is unaltered with change of pH from 6.8 to 5.7. 相似文献
17.
18.
Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath 总被引:1,自引:0,他引:1
Soluble extracts of Methylococcus capsulatus (Bath) that readily oxidise methane to methanol will also oxidise ammonia to nitrite via hydroxylamine. The ammonia oxidising activity requires O2, NADH and is readily inhibited by methane and specific inhibitors of methane mono-oxygenase activity. Hydroxylamine is oxidised to nitrite via an enzyme system that uses phenazine methosulphate (PMS) as an electron acceptor. The estimated K
mvalue for the ammonia hydroxylase activity was 87 mM but the kinetics of the oxidation were complex and may involve negative cooperativity.Abbreviations PMS
Phenazine methosulphate
- NADH
nicotinamide adenine dinucleotide, reduced form
-
K
m
Michaelis constant
- NO
2
-
nitrite
- NH2OH
hydroxylamine 相似文献
19.
A. Fjellbirkeland Hans Kleivdal Carsten Joergensen Helle Thestrup Harald B. Jensen 《Archives of microbiology》1997,168(2):128-135
Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed
by electron microscopy and by the content of β-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins
with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble
membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties
in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated
as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes,
this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl.
Received: 20 December 1996 / Accepted: 11 April 1997 相似文献
20.