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1.
A simple method for purification and crystallization of primary alcohol dehydrogenase (EC 1.1.99.8) is reported. The purification procedures consisted of four steps: protamine sulfate treatment, ammonium sulfate fractionation, passage through a column of DEAE-cellulose at pH 8.0 and Sephadex G-200 gel filtration. Crystallization was performed by the addition of ammonium sulfate at 65 % saturation with an overall yield of 39 %. The crystalline enzyme had an isoelectric point of pH 7.38 and a sedimentation coefficient 8.44s. A molecular weight of 128,000 was estimated, and the enzyme consisted of two subunits each having a molecular weight of 62,000. The enzyme showed an affinity toward the lower primary alcohols, methanol to n-pentanol. Formaldehyde was also oxidized by the crystalline enzyme. The Km values for methanol and formaldehyde were found to be 20 μm and 70 μm, respectively. Ammonium ions were required for enzyme activity.  相似文献   

2.
The increasing number of species for which a full genome sequence is available offers rich pickings for geneticists, but comparative analysis and assembly of information gathered across species does not always lead to answers about the function of a particular gene. This paper aims to place the invertebrate model system--the fly Drosophila melanogaster--into this playing field and to discuss how the organism arrived at its position in functional genetic analysis. Indeed, despite the wealth of knowledge on how a fly lives, breathes and flies, this organism is likely to remain a player in the analysis of biological, disease and pharmaceutical processes. The fast genetics Drosophila offers, combined with a well-annotated genome and a wealth of techniques facilitating gene function discovery, will ensure its place in functional genomics for some time to come. Although the fly cannot speak, it certainly can tell a tale.  相似文献   

3.
An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative deamination of various aromatic amines as well as some aliphatic amines to a lesser extent. An artificial electron acceptor such as phenazine methosulfate was required for the catalysis. The molecular weight determined by sedimentation equilibrium was 103,000 and the molecule seemed to be composed of two pairs of two nonidentical subunits (Mr 46,000 and 8000). The enzyme had a dull yellow-green color with an absorption maximum at 445 nm and this chromophore appeared to be involved in the catalytic action of the enzyme.  相似文献   

4.
N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.  相似文献   

5.
We have analyzed Ca2+ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca2+ current that is not affected by L-type Ca2+ channel blocker (PN 200-110), N-type Ca2+ channel blocker (-conotoxin GVIA) or P-type Ca2+ channel blocker (Agatoxin IVA) but is blocked by either Cd2+ or Ni2+. This pharmacological profile most closely resembles that of T-type Ca2+ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca2+ channel window current. We suggest that Ca2+ overload due to abnormal voltage-dependence of transient Ca2+ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca2+ channel currents, Ni2+ significantly reduced cell death in cell lines transfected with polyGln-expanded AR.  相似文献   

6.
A sarcosine dehydrogenase was purified to homogeneity from cell free extract of Pseudomonas putida aerobically grown in a medium containing creatinine or betaine as the carbon and nitrogen sources. The enzyme catalyzed dehydrogenation of N-methyl derivatives of some amino acids but was inert toward dimethylglycine, betaine and choline. Phenazine methosulfate, 2, 6-dichlorophenol indophenol, methylene blue, meldora blue, nile blue and potassium ferricyanide served as electron carriers. The maximal activity was observed at pH 8.0–9.0. The Km and Kmax values for sarcosine were 29 mm and 1.2 μmol/min/mg, respectively. The molecular weight was estimated to be about 170,000, presumably composed of four sub-units. Spectrophotometric and fluorometric analyses indicated that the enzyme was a flavoprotein.  相似文献   

7.
Dienelactone hydrolase (EC 3.1.1.45) from Pseudomonas sp. B13 has been crystallized in a form suitable for high resolution x-ray diffraction study. The crystals are orthorhombic, the space group being P212121, with unit cell dimensions a = 48.9 A, b = 71.2 A, and c = 77.5 A. There appears to be 1 molecule in the asymmetric unit.  相似文献   

8.
9.
The dimeric L -2-haloacid dehalogenase from Pseudomonas sp. YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n-propanol. The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 Å, b = 62.78 Angst; c = 50.84 Å, and β = 122.4°, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 Å resolution.  相似文献   

10.
Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.  相似文献   

11.
An NADP-specific glutamate dehydrogenase [L-glutamate: NADP+ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (Km 44 μM), 2-oxoglutarate (Km 3.13 mM), NADP+ (Km 29 μM), and L-glutamate (Km 6.06 mM). The Km for NH4Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.  相似文献   

12.
Proteolysis of polyglutamine-expanded proteins is thought to be a required step in the pathogenesis of several neurodegenerative diseases. The accepted view for many polyglutamine proteins is that proteolysis of the mutant protein produces a “toxic fragment” that induces neuronal dysfunction and death in a soluble form; toxicity of the fragment is buffered by its incorporation into amyloid-like inclusions. In contrast to this view, we show that, in the polyglutamine disease spinal and bulbar muscular atrophy, proteolysis of the mutant androgen receptor (AR) is a late event. Immunocytochemical and biochemical analyses revealed that the mutant AR aggregates as a full-length protein, becoming proteolyzed to a smaller fragment through a process requiring the proteasome after it is incorporated into intranuclear inclusions. Moreover, the toxicity-predicting conformational antibody 3B5H10 bound to soluble full-length AR species but not to fragment-containing nuclear inclusions. These data suggest that the AR is toxic as a full-length protein, challenging the notion of polyglutamine protein fragment-associated toxicity by redefining the role of AR proteolysis in spinal and bulbar muscular atrophy pathogenesis.  相似文献   

13.
To put forward BDH from Pseudomonas aeruginosa’s enzymatic properties, we report a two-step purification of BDH and its gene sequencing allowing the investigation of its structural properties. Purification of BDH was achieved, using ammonium sulfate fractionation and Blue Sepharose CL-6B affinity chromatography. SDS–PAGE analysis reveals a MM of 29 kDa, whereas the native enzyme showed a MM of 120 kDa suggesting a homotetrameric structure. BDH encoding gene sequence shows a nucleotide open reading frame sequence of 771 bp encoding a 265 amino acid residues polypeptide chain. The modeling analysis of the three dimensional structure fits with the importance of amino acids in the catalysis reaction especially a strictly conserved tetrad. Amino-acid residues in interaction with the coenzyme NAD+ were also identified.  相似文献   

14.
Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on β-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (α2β2) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the α-subunit (42.3-kDa subunit) and the β-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the β-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and β-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 °C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, β-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 μM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 °C for one month at least in phosphate buffer (pH 7.0).  相似文献   

15.
目的:为了探讨3株Pseudomonas sp.对吡啶降解存在多样性.方法:基于16S rRNA和ISR序列分析,对3株分离菌株进行初步鉴定,进而通过Touch -Down PCR,对3株细菌降解吡啶的多样性进行分析.结果:3株细菌XJUHX -1、XJUHX - 12和XJUHX - 16初步鉴定为Pseudomonas,3株实验菌株的部分降解基因的扩增条带有差异.结论:同属的3株 Pseudomonas在吡啶降解上存在多样性.  相似文献   

16.
Glutaryl-7-aminocephalosporanicacid acylase from Pseudomonas sp. GK16 produces glutaryl-7-aminocephalosporanic acid, a key intermediate for the synthesis of cephem antibiotics. Sequence alignment suggests that the enzyme may belong to the N-terminal nucleophile hydrolase superfamily including penicillin G acylase. The enzyme is an (alphabeta)(2) heterotetramer of two nonidentical subunits. These subunits are derived from a nascent precursor polypeptide that is cleaved proteolytically through a two-step autocatalytic process upon folding. The enzyme has been crystallized using the vapor diffusion method. A bipyramidal crystal form was obtained from a solution containing polyethylene glycol (MW 3350) and calcium chloride. Complete diffraction data sets have been collected up to 2.8 A resolution. The crystal is tetragonal with the space group P4(1)2(1)2 or P4(3)2(1)2 and the unit cell parameters are a = b = 73.5 A, c = 380.3 A. Considerations of the possible values of V(m) account for the presence of a tetramer in the asymmetric unit.  相似文献   

17.
假单胞菌WBC—3甲基对硫磷水解酶性质的初步研究   总被引:12,自引:0,他引:12  
从最近分离到的有机磷农药降解菌Pseudomonas sp.WBC—3中获得了甲基对硫磷水解酶(Methyl parathion hydrolase,MPH,EC3.1.8.3)。该酶在48h的培养物中分布比例分别为:上清液2.1%,胞内86.2%和胞间质11.7%,说明MPH为胞内酶。经过CM—sepharose Fast Flow阳离子交换层析,获得电泳纯的酶。SDS—PAGE和凝胶过滤层析表明,该酶为单体蛋白,分子量约为34kD。动力学分析显示该酶为非特异性有机磷降解酶,但最适底物为甲基对硫磷。在pH9~12范围,酶表现较高活力水平,最高活力的反应温度为40℃。根据各类金属离子和鳌合剂对酶活的影响,推测MPH为金属酶。  相似文献   

18.
The distribution coefficients of glucose, maltose, and maltotriose on cation-exchange resin in Na+ form with a divinylbenzene content of 4% were determined in the temperature range of 5 to 60°C. The coefficients increased with decreasing temperature. The temperature dependence of the distribution coefficient was analyzed based on the swelling pressure of the resin.  相似文献   

19.
NAD+-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2–10). The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane. Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer. The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree. Thus, the modified enzyme mainly functions in the form of monomer and octamer. The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.  相似文献   

20.
The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD+-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α2 structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The Km values for coniferyl aldehyde and NAD+ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH2-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD+-dependent aldehyde dehydrogenases from different sources.  相似文献   

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