首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α2β2γ2 structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg2+ ions (with a total loss of enzyme activity at 0.01 mM Hg2+) and Cu2+, Zn2+, and Ni2+ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).  相似文献   

2.
The purpose of this study was to characterize the physical properties of cytochromec oxidase from rat liver. The enzyme was extracted from isolated mitochondria with nonionic detergents and further purified by ion-exchange chromatography on DEAE Bio-Gel A. The purified enzyme contained 9.64 nmol heme a/mg protein and one iron atom plus one copper atom for each heme a. The specific activity of the final preparation was 146 µmol of ferrocytochromec oxidized/min · mg protein, measured at pH 5.7. The spectral properties of the enzyme were characteristic of purified cytochrome oxidase and indicated that the preparation was free of cytochromesb, c, andc 1. In analytical ultracentrifugation studies, the enzyme sedimented as a single component with anS 20,w of5.35S. The Stokes radius of the enzyme was determined by gel filtration chromatography and was equal to 75 Å. The molecular weight of the oxidase calculated from its sedimentation coefficient and Stokes' radius was 180,000, indicating that the active enzyme contained two heme a groups. The purified cytochrome oxidase was also subjected to dodecyl sulfate-polyacrylamide gel electrophoresis in order to determine its components. The enzyme was resolved into five polypeptides with the molecular weights of I, 27,100; II, 15,000; III, 11,900; IV 9800; and V, 9000.  相似文献   

3.
When Frateuria species ANA-18 was grown on aniline, two catechol 1,2-dioxygenases (CD I and CD II, EC 1.13.11.1) were found in a cell-free extract of the strain. CD I and CD II were separated from each other by DE-52 chromatography and purified to homogeneity by successive column chromatography. The molecular weights of CD I and CD II were 38,000 and 36,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CD I contained 2 mol of sulfhydryl groups per mol of protein, but CD II did not have cysteine or cystine residues. CD I was more stable on the acidic side and against heating than CD II. The activity of CD I was inhibited by AgN03, HgCl2, iodoacetic acid and p-chloromercuribenzoic acid, but that of CD II was not inhibited or was less affected by them because of the lack of sulfhydryl groups. CD I exhibited a high activity for the extradiol cleavage of 3-methylcatechol, and the ratio of intradiol to extradiol activity was 100 :120.  相似文献   

4.
Mucous glycoproteins were isolated by agarose gel filtration from nonpurulent tracheobronchial secretions and purulent sputum which had been reduced, carboxymethylated and, in the case of purulent secretions, treated with deoxyribonuclease. The solubilized and purified glycoproteins were fractionated on diethylaminoethyl cellulose into two major (I, II) and two minor (Ia, III) blood group active components. Components I and II had similar carbohydrate and amino acid compositions which were typical for human blood group substances. These two components did differ in several respects. Component I contained 1.4–2.6% sulfate and did not inhibit influenza virus hemagglutination while component II contained 7.1–7.8% sulfate and was a potent inhibitor of virus hemagglutination. Component II also migrated more rapidly on sodium dodecyl sulfate-3.3% acrylamide gel electrophoresis. Components I and II in purulent secretions displayed only minor compositional differences from their counterparts in nonpurulent secretions. Component II was more abundant in two sputum samples from subjects with cystic fibrosis than in purulent bronchitic secretions or in nonpurulent secretions.  相似文献   

5.
6.
Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca2+ or Mg2+. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca2+ and Mg2+. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca2+ and Mg2+, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.  相似文献   

7.
Extracts of Pseudomonas sp. strain CBS3 grown with 4-chlorobenzoate as sole carbon source contained an enzyme that converted 4-chlorobenzoate to 4-hydroxybenzoate. This enzyme was shown to consist of three components, all necessary for the reaction. Component I, which had a molecular weight of about 3,000, was highly unstable. Components II and III were stable proteins with molecular weights of about 86,000 and 92,000.  相似文献   

8.
Extracts of Pseudomonas sp. strain CBS3 grown with 4-chlorobenzoate as sole carbon source contained an enzyme that converted 4-chlorobenzoate to 4-hydroxybenzoate. This enzyme was shown to consist of three components, all necessary for the reaction. Component I, which had a molecular weight of about 3,000, was highly unstable. Components II and III were stable proteins with molecular weights of about 86,000 and 92,000.  相似文献   

9.
Aspergillus flavus grown in a liquid medium containing pectin as the sole carbon source produced extracellular enzymes which degraded the 1,4-α-d-glycosidic bonds of pectin. The products of degradation were characteristic of substances produced by transeliminase. Synthesis of this enzyme was repressed by the addition of sucrose, glucose, fructose and maltose. The crude enzyme was partially purified by a combination of ultrafiltration and ammonium sulfate precipitation. The partially purified enzyme was separated by molecular exclusion chromatography into three components A, B and C, with molar masses ranging from 13.2 to 64 kDa. Only fraction B exhibited enzymic activity and further fractionated by ion-exchange chromatography into four components I–IV. Among these components, only fractions I and II possessed transeliminase activity. Both fractions had an optimum activity at pH 8.5 and 35°C, and were stimulated by Ca2+, Mg2+, Na+ and K+ but inhibited by EDTA and DNP. The apparentK m for the degradation of pectin by fractions I and II were 6.2 and 8.0 g/L, respectively.  相似文献   

10.
Two isoforms of laccase produced from the culture supernatant of Pycnoporus sanguineus were partially purified by phenyl-Sepharose chromatography. Molecular masses of the enzymes were 80 kDa (Lac I) and 68 kDa (Lac II). Optimum activity of Lac I was at pH 4.8 and 30 °C, and Lac II was at pH 4.2 and 50 °C over 5 min reaction. The Km values of enzymes toward syringaldazine were 10 μm (Lac I) and 8 μm (Lac II). Sodium azide inhibited Lac I (85%) and Lac II (75%) activities. Revisions requested 30 November 2005; Revisions received 26 January 2006  相似文献   

11.
Endogenous gibberellins from floating plants and in vitro induced turions of Wolffiella floridana (Lemnaceae) were extracted and partially purified. Gibberellin-like activity was detected in two zones of the chromatogram corresponding to Rf 0-0.1 and Rf 0.4-0.5 by dwarf pea bioassay. The active compounds in the two zones have been referred to in this paper as factor I and factor II respectively. There were quantitative differences in the gibberellin-like substances of floating plants and turions. The floating plants contained more of factor I but less of factor II as compared to the turions. The chromatographic behavior of factor I was similar to either gibberellin A1 and A3 while the identity of factor II was uncertain.  相似文献   

12.
Endoglucanase ofRuminococcus sp. is composed of seven active protein components when chromatographed on an ion exchange column (Q-Sepharose). Component I (endoglucanase A) did not bind to the column and was purified to homogeneity by molecular sieve chromatography. It had a mol. wt. of 22 000. Component II was fractionated into two active protein peaks (endoglucanase B and C) having mol. wt. of 225 000 and 10 000. The endoglucanase A had high affinity for CMC (Km 8 mg/ml). The temperature optimum of all three endoglucanase was between 40–45°C. The gene encoding for endolucanase activity was cloned inE. coli HB101 with pBR322. A 4.3 kilobaseBamH1 fragment encoding endoglucanase was hybridized toRuminococcus chromosomal DNA.  相似文献   

13.
Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O2- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.  相似文献   

14.
Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (Ki) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human neutrophil elastase inhibitors described in a mollusk.  相似文献   

15.
Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 μmol H2 oxidized min–1 (mg protein)–1 with benzyl viologen as electron acceptor and an apparent K m value for H2 of 341 μM. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively. SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained 15.6 ± 1.7 mol Fe, 11.4 ± 1.4 mol S2–, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)+. The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases. Washed membranes catalyzed a H2-dependent cytochrome b reduction at a rate of 0.18 nmol min–1 (mg protein)–1. Received: 7 September 1995 / Accepted: 4 December 1995  相似文献   

16.
Ferredoxin was purified to apparent homogeneity from cell extracts of the homoacetogen Peptostreptococcus productus (strain Marburg). The yield was 70 g ferredoxin per g wet cells of P. productus. The UV-vis spectrum exhibited characteristics of a typical clostridial ferredoxin spectrum with a molar extinction coefficient 385 of 30000 M-1 cm-1 and an A385/A280 ratio of 0.76. The molecular weight Mr was near 5700 as calculated from the amino acid composition. The protein contained per mol 9.9 mol iron, 8.2 mol acid-labile sulfide, and near 7 mol cysteine indicating the presence of two 4 Fe/4 S clusters. The redox potential was determined to be-410 mV. The purified ferredoxin was reduced with carbon monoxide by the carbon monoxide dehydrogenase from crude extracts and by the partially enriched enzyme of P. productus.  相似文献   

17.
The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl2, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.  相似文献   

18.
Three distinct forms of -glucosamine 6-P (Gm 6-P):N-acetyltransferases (EC 2.3.1.4) were partially purified from human placental homogenates by carboxy methyl-Sephadex chromatography. Purification of forms I and II were 13.5-fold, while that of form III was 114-fold. All three forms had a pH optimum value of 9.7 in glycine–NaOH buffer. Enzymes II and III had a Km value for Gm 6-P of 3.0 mM, which was less than half of that observed for form I (7.1 mM). The corresponding Km values for acetyl CoA were 0.157 (form I), 0.187 (form II) and 0.280 mM (form III), respectively. Activities of all three forms were inhibited at high concentrations of either substrate. These enzymes were inhibited from 82 to 92% by 2.5 mM p-chloromercuribenzoate. The inhibition was largely reversible by inclusion of 2.5 mM dithiothreitol in the incubation mixtures. There was no requirement for divalent cations, as demonstrated by lack of inhibition of enzyme activity by ethylene diamine tetraacetate. The results are discussed in terms of differences among the enzyme properties of human placental, rodent and porcine liver forms.  相似文献   

19.
Germinating barley grown on an artificial medium was exposed to75Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and75Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.  相似文献   

20.
The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H2 plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号