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1.
An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (Km = 5.5 × 10−6 M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (Ki = 1.1 × 10−8 M) and leupeptin (Ki = 3.9 × 10−8 M) but was not inhibited with Nα-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.  相似文献   

2.
The fructose-1,6-P2 (FDP) phosphatase, (FDPase) and FDP aldolase fromPseudomonas putida were partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. Michaelis-Menten kinetics were observed with, respect to FDP in both FDPase and FDP aldolase. TheK m for FDP at pH 8.0 was 1.2×10−5M for FDPase and 3.0×10−5M for FDP aldolase. The specific activities of these two enzymes (assayed under optimal conditions in cell-free extracts ofP. putida grown ond-fructose), as well as their kinetic properties, are consistent with the suggestion that during growth ond-fructose most, of the FDP generated is converted to fructose-6-P (F-6-P), which is subsequently utilized via the Entner-Doudoroff pathway (EDP).  相似文献   

3.
Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK i =1.66±3.26×10−3, 1.92±1.63×10−4 and 2.16±3.79×10−4) and binding constant of cadmium metal ions (pK M1=1.99±2.45×10−3 and pK M2=1.67±4.08×10−3) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK f) constant is 2.04±2.1×10−5 suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10−3± 2.10×10−3, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.  相似文献   

4.
Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na+ and/or K+ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg++ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg++, Mn++ and Ca++ ions exerting the most positive effect. Determination of the specificity constants (k cat/K m) clearly indicated that BbgI (6.11 × 104 s−1 M−1), BbgIII (2.36 × 104 s−1 M−1) and especially BbgIV (4.01 × 105 s−1 M−1) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 104 s−1 M−1) than lactose (1.48 × 103 s−1 M−1). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.  相似文献   

5.
From the blue seed coats ofOphiopogon jaburan, a new flavonol glycoside was isolated as needles and determined to be kaempferol 3-O-β-d-galactoside-4′-O-β-d-glucoside (OK-2) by UV and NMR spectral analyses. OK-2 and kaempfrol 3, 4′-di-O-β-d-glucoside (OK-1), which was detected previously, in the blue seed coat were present in a molar ratio of about 13:7. OK-2 was newly found as a factor causing the blueing effects on ophionin which is a main anthocyanin in the blue seed coats. The mixture of 4.8×10−3 M OK-2 and 2.5×10−3 M ophionin in Mcllvaine's buffer solution (pH 5.6) showed stable blue color, and the absorption spectrum of the mixture showed two absorption peaks and a shoulder in visible reasion, coinciding with that of the fresh blue seed coat. The effect of ophionin and OK-2 co-pigmentation on the blue color of seed coat ofO. jaburan was discussed.  相似文献   

6.
Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p Gly ) was 2.3 ± 0.23 × 10−6 cm sec−1 with MIP vs. 0.92 ± 0.086 × 10−6 cm sec−1 in control oocytes. The p Gly of MIP was independent of concentration from 5 × 10−5 to 5 × 10−2 m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg++, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min−1 cell−1) without changing the binding of glycerol to the kinase (K M ∼ 10 μm). Received: 23 May 1997/Revised: 4 August 1997  相似文献   

7.
Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58×10−6 m and a Vmax of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8×10−8 m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the KM for putrescine, 50.3×10−6 m. The K1 of the ABS putrescine oxidase for aminoguanidine was 41×10−6 m. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.  相似文献   

8.
Characteristics for the up-regulated response in the concentration of intracellular calcium ion ([Ca2+] i ) and in the sodium ion (Na+) current by serotonin (5-HT) were investigated in differentiated neuroblastoma × glioma hybrid NG108-15 (NG) cells. The results for the changes in [Ca2+] i by 5-HT were as follows, (1) The 5-HT-induced Ca2+ response was inhibited by 3 × 10−9 M tropisetron (a 5-HT3 receptor blocker), but not by other types of 5-HT receptor blockers; (2) The 5-HT-induced Ca2+ response was mainly inhibited by calciseptine (a L-type Ca2+ blocker), but not by other types of Ca2+ channel blockers or 10−7 M TTX (a voltage-sensitive Na+ channel blocker); (3) When the extracellular Na+ was removed by exchange with choline chloride or N-methyl-d-glucamine, the 5-HT-induced Ca2+ response was extremely inhibited. The results for the 5-HT-induced Na+ current by the whole cell patch-clamp technique were as follows, (1) The 5-HT-induced Na+ current in differentiated cells was significantly larger than that in undifferentiated cells; (2) The ED50 value for 5-HT-induced Na+ current in undifferentiated and differentiated cells was almost the same, about 4 × 10−6 M each other; (3) The 5-HT-induced Na+ current was completely blocked by 3 × 10−9 M tropisetron, but not by other 5-HT receptor antagonists and 10−7 M TTX. These results suggested that 5-HT-induced Ca2+ response in differentiated NG cells was mainly due to L-type voltage-gated Ca2+ channels allowing extracellular Na+ to enter via 5-HT3 receptors, but not through voltage-gated Na+ channels.  相似文献   

9.
Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK m andV max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min−1 mg−1, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min−1 mg−1, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn2+. However, excessive Zn2+ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.  相似文献   

10.
The presence of an electrogenic H+-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (V t ) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 × 10−7 m bafilomycin or 4.6 × 10−8 m concanamycin were added. No significant increase in lumen-negative V t upon perfusion with these inhibitors as compared to control, was observed as well as when 10−3 m amiloride, 10−5 m benzamil or 3 mm Ba2+ were perfused alone or in combination. The effect of an inhibition of electrogenic H+ secretion, i.e., increase in lumen-negative V t by 2–4 mV, was observed only when Cl channels were blocked by 10−5 m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm−2.sec−1 as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H+ ATPase in the establishment of cortical late distal tubule V t is minor in physiological conditions, but can be demonstrated after blocking Cl channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H+ secretion in this segment is reduced by a Cl channel blocker, supporting coupling of H+-ATPase with Cl transport. Received: 6 July 1996/Revised: 27 December 1996  相似文献   

11.
Three methanol-oxidizing bacteria were isolated by enrichment culture technique from soil. Two of them werePseudomonas spp. The third one was obligate methylotroph. Their growth characteristics have been described. Cell-suspension experiments withPseudomonas RJ1 suggest that methanol-, formaldehyde-, and formate-oxidizing enzymes were present. Formate-oxidizing enzyme was detected only fromPseudomonas RJ1. It had a pH optimum of 7.0 and required nicotinamide adenine dinucleotide (NAD) for activity. Cyanide at 1 × 10−5 m concentration inhibited the enzyme activity completely.  相似文献   

12.
Yao YF  Weng YM  Hu HY  Ku KL  Lin LL 《The protein journal》2006,25(6):431-441
A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K m and V max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10−3 mM min−1, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM L-Gln, 40 mM ethylamine, and 1.04 U His6-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.  相似文献   

13.
An endoglucanase (1, 4-β-d glucan glucanohydrolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley β-glucan, xyloglucan and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN1 following ion-exchange and hydrophobic interaction chromatography and gel filtration. The purified enzyme (40-fold) that apparently lacked a cellulose-binding domain showed a specific activity of 60 μmol mg−1 protein−1 against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60°C and pH 4.0, and was stable over a broad range of pH (3.0–5.0) at 50°C. The endoglucanase activity was positively modulated in the presence of Cu2+, Mg2+, Ca2+, Na+, DTT and mercaptoethanol. Endoglucanase exhibited maximal turn over number (K cat) and catalytic efficiency (K cat/km) of 19.11 × 105 min−1 and 29.7 × 105 mM−1 min−1 against barley β-glucan as substrate, respectively. Hydrolysis of CMC and barley β-glucan liberated cellobiose, cellotriose, cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, however, apparently yielded positional isomers of cellobiose, cellotriose and cellotetraose as well as larger oligosaccharides.  相似文献   

14.
Two trypsin isoforms (GT-A and GT-B) from the grass carp (Ctenopharyngodon idellus) intestine were isolated and purified. SDS-PAGE electrophoresis showed that GT-A and GT-B had relative molecular masses of 30,740 and 26,400, respectively. Enzyme activity was inhibited by three organic trypsin inhibitors but not by EDTA. They had optimal pH of 8.0 and 8.5, and optimal temperatures of 38.5 and 44.0°C, respectively, when hydrolyzing N–benzoyl-l-arginine ethyl ester·HCl (BAEE). They lost 95.8 and 93.7% of their activities, respectively, after heating for 20 min at 65°C. Their thermal denaturation temperatures, respectively, were 66.3 and 67.3°C. GT-A has a Km value of 21.2 μM and a Vmax of 2.0 × 103 min−1, and GT-B has a Km value of 31.7 μM and a Vmax of 3.3 × 103 min−1. Their physiological efficiencies were 94.3 and 105.3 μM−1 min−1, respectively. The Arrhenius activation energies of GT-A and GT-B were 4.16 and 4.38 kcal/mol, respectively. The activities of GT-A and GT-B were not activated by Ca2+, but their thermostability was improved in the presence of Ca2+. Enzyme activity was reduced in presence of Zn2+, Cu2+, Hg2+ and Al3+. Thermal stabilities of GT-A and GT-B were intermediate between Arctic and tropical fish species, and consistent with the wide range of water temperatures to which grass carp are exposed in most provinces of China.  相似文献   

15.
The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: Km (Ala)=1.53 mM, Km (2-oxoglutarate)=0.18 mM, kcat=124.6 s−1, kcat/Km=8.1 × 104 M−1·s−1 of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated Km values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.  相似文献   

16.
Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K 0 = (3.9 ± 0.4) × 10−4 and (1.3 ± 0.1) × 10−5 M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K h = (4.3 ± 0.4) × 10−3 and (1.2 ± 0.1) × 10−4 M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H 0 = (1.9 ± 0.2) × 10−8 M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H d = (2.1 ± 0.2) × 10−7 M]. In the absence and presence of CO2, the values of the second-order rate constant (l on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 105 and 4.3 × 105 M−1 s−1, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO2. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.  相似文献   

17.
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml−1). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K m and K cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 105 min−1, respectively. The catalytic efficiency (K cat /K m) was 7.23 × 108 min−1 M−1. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.  相似文献   

18.
The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10−6 m BAP, 4.6 × 10−6 m KIN, or 4.9 × 10−6 m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10−8 m BAP, 4.6 × 10−8 m KIN, 4.5 × 10−8 m ZEA, or 4.9 × 10−8 m 2iP) promoted embryo growth. TDZ at 9.9 × 10−9 m, 9.9 × 10−8 m, or 9.9 × 10−7 m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10−7 m ZEA. Received August 1, 1997; accepted February 19, 1998  相似文献   

19.
A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml−1, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml−1 only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml−1 in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its Km and Vmax were 14 μm and 3.8 nmol min−1 mg−1 respectively. The enzyme activity was enhanced by Cu2+ and Ni2+ and inhibited by Hg2+. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005  相似文献   

20.
Chloride (Cl) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl current activated by 10−5 m forskolin, 10−3 m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10−6 m phorbol 12-myristate 13-acetate (PMA), 10−3 m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10−7 m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br > Cl≫ I > glutamate. This current was inhibited by 10−3 m diphenylamine-2-carboxylate (DPC) and 10−4 m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10−3 m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl current blocked by DIDS. To determine the exact location of the Cl conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl removal from the apical solution induced a Cl efflux which was stimulated by 10−5 m forskolin, 10−7 calcitonin and inhibited by 10−5 m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br influx through the Cl pathway. Forskolin and calcitonin had no effect on the basolateral Cl permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995  相似文献   

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