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1.
The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min?1 g?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics. 相似文献
2.
Inversion of sucrose with β-
-fructofuranosidase (EC 3.2.1.26) immobilized by the ionic bond on bead DEAHP-cellulose has been studied under flow conditions. Under these conditions, the inversion of sucrose is affected by the concentration and flow rate of the substrate and by the reaction temperature. The effect of substrate concentration on the reaction was investigated in the range 19.5–64.2 wt %; the effect of flow rate was examined in the range 0.25–5.57 g solution per min, and the temperature range used was 25–50°C. It was found that the activities of immobilized β-
-fructofuranosidase in stirred and flow reactors were the same. The lower activities of β-
-fructofuranosidase in the case of concentrated solutions, and of immobilized β-
-fructofuranosidase compared with the native enzyme are attributed to more difficult diffusion through the beads of the ion exchanger, especially of the strongly viscous substrate. A long-term investigation of the enzyme activity over a period of three months demonstrated the stability of the β-
-fructofuranosidase immobilized by the ionic bond on bead DEAHP-cellulose; the half-life of the enzyme was 215 days. It was also found that the immobilization of the enzyme on a carrier was more effective under flow conditions, i.e. through an ion exchanger in the column, than under the equilibrium conditions of a stirred reactor. 相似文献
3.
A hydrogen peroxide permselective membrane with asymmetric structure was prepared and
-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized
-glucose oxidase membrane was 0.34 units cm−2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized
-glucose oxidase membrane was 1.6 × 10−3 mol l−1 and that of free enzyme was 4.8 × 10−2 mol l−1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized
-glucose oxidase membrane. The enzyme electrode responded linearly to
-glucose over the concentration 0–1000 mg dl−1 within 10 s. When the enzyme electrode was applied to the determination of
-glucose in human serum, within day precision (CV) was 1.29% for
-glucose concentration with a mean value of 106.8 mg dl−1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized
-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of
-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months. 相似文献
4.
Jeak Ling Ding Yong Zhu Bow Ho 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,759(2):237-246
Synthetic peptide S3Δ has high affinity for bacterial endotoxin or lipopolysaccharide (LPS). Under tested conditions of pH 5–9 and 0–0.4 M NaCl, the affinity constant, KD ranged from 2·10−6 to 2·10−9 M−1. A novel affinity matrix based on peptide S3Δ was developed for removal of LPS from solutions such as: water; buffers with a wide range of ionic strength and pH; medium for cell culture; and protein solutions under optimized conditions. At a starting LPS of ≈100 EU/ml, a post-purification level below 0.005 EU/ml was achieved. 相似文献
5.
S.U. Jadhav S.D. Kalme S.P. Govindwar 《International biodeterioration & biodegradation》2008,62(2):135-142
Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl−1) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl−1 molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l−1 concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l−1 concentration.
The scientific relevance of the paper
The azo dye Methyl red (100 mg l−1) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants. 相似文献6.
Ashok Kumar Patel Vijay Kumar Singh Ravi Prakash Yadav A.J.G. Moir Medicherla V. Jagannadham 《Phytochemistry》2009,70(10):1210-1216
A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters Km, Vmax, Kcat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10−8 mol min−1 μg enzyme−1, 29.0 s−1 and 58.0 mM−1 s−1 respectively. The extinction coefficient was estimated as 20.56 M−1 cm−1. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields. 相似文献
7.
Δ2-Thiazoline-2-carboxylate, the product of the suspected physiological reaction catalyzed by
-amino acid oxidase, is stable to hydrolysis at 37°C and pH 7 or above, but it hydrolyzes readily at pH 5 or below to give a mixture of N- and S-oxalylcysteamines; the N-oxalyl derivative predominates at pH's above 1 while the S-oxyalyl compound is the major product at high acidities. The pH-rate profile looks like the superposition of two bell-shaped curves. The initial increase in the rate as the pH is lowered is controlled by a pKa of 3.95 and from pH 1 to 3 the rate is relatively constant (k = 6.7 × 10−4s−1 at 37°C and ionic strength 0.5
). Below pH 1 the rate increases again to a maximum in 1
HCl and then decreases in more highly acidic solutions. The rate of conversion of S-oxalylcysteamine to N-oxalylcysteamine is inversely proportional to the hydrogen ion concentration from pH 3 to 5 but becomes largely independent of pH from pH 1 to 2. In the pH-independent region the rate is comparable with that observed by others for S-acetylcysteamine but in the pH-dependent region the rate is 20 to 25 times faster for the oxalyl derivative than for the acetyl compound. At pH 1, N-oxalylcysteamine is partially converted to the S-oxalyl derivative but the rate of hydrolysis (k = 1.0 × 10−5s−1 at 37°C) to cysteamine and oxalate of this partially equilibrated system occurs at a comparable rate. The results of this investigation are rationalized in terms of what is known about other thiazoline hydrolyses and intramolecular S to N acyl migrations. The main differences in the present case are presumably due to the fact that thiazoline-2-carboxylate can undergo hydrolysis by two reaction manifolds, one with the carboxyl unprotonated and the other with it protonated. The relevance of these results to possible reactions of thiazoline-2-carboxylate in vivo is briefly considered. 相似文献
8.
The extracellular carboxymethyl cellulase (CSCMCase) from the yeast, Cryptococcus sp. S-2, was produced when grown on cellobiose. It was purified to homogeneity from the supernatant by ultrafiltration, DEAE-5PW anion exchange column and TSK-Gel G3000SW gel filtration. The purified enzyme was monomeric protein with molecular mass of approximately 34 kDa. The optimum temperature and pH for the action of the enzyme were at 40–50 °C and 3.5, respectively. It was stable at pH range of 5.5–7.5 and retained approximately 50% of its maximum activity after incubating at 90 °C for 1 h. Moreover, it could able to hydrolyze carboxymethyl cellulose sodium salt higher than insoluble cellulose substrate such as Avicel, SIGMACELL® and CM cellulose. Due to its action at acidic pH and moderately stable at high temperature, the gene encoding carboxymethyl cellulase (CSCMCase) was isolated and improved the enzyme yield by high cell-density fermentation of Pichia pastoris. The CSCMCase cDNA contains 1023 nucleotides and encodes a 341-amino acid. It was successfully expressed under the control of alcohol oxidase I promoter using methanol induction of P. pastoris fermentation in a 2L ABLE bioreactor. The production of the recombinant carboxymethyl cellulases was higher than that from Cryptococcus sp. S-2 of 657-fold (2.75 and 4.2 × 10−3 mg protein L−1, respectively) indicating that the leader sequence of CSCMCase has been recognized and processed as efficiently by P. pastoris. Furthermore, the recombinant enzyme was purified in two-step of ultrafiltration and hydrophobic interaction chromatography which would be much more convenient for large-scale purification for successful industrial application. 相似文献
9.
Sandeep Kumar Alka Dwevedi Arvind M. Kayastha 《Journal of Molecular Catalysis .B, Enzymatic》2009,58(1-4):138-145
The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol−1, 5.02 kcal mol−1, 6.45 kcal mol−1 for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in Km value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of Vmax for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 102 μmol NH3 min−1 mg−1 protein, 2.65 × 102 μmol NH3 min−1 mg−1 protein and 2.85 × 102 μmol NH3 min−1 mg−1 protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer®. The immobilization studies have a potential role in haemodialysis machines. 相似文献
10.
Benjamin J. Danzo Vathsala Krishnamurthy Barbara C. Eller 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,500(2):310-321
Macromolecular binding components for [3H]estradiol-17β are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4–5 S and the other had a sedimentation coefficient of 8–9 S. The two components differed from each other regarding steroid specicity and various physiocochemical parameters. [3H]-estradiol binding to the 4–5 S component was not inhibited by estrogens, 5α-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appera to be saturable and lavel was rapidly stripped from it by cahrcoal. Estradiol bindng to the 8–9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4–5 S moiety. The specific binding protein has a Kd of 3.05 · 10−10 M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incbuation of [3H]estradiol with mature male liver cytosol at 0–5°C polar metabolites of estradiol are produced. 相似文献
11.
Yangyang Jiang Chen Guo Hansong Xia Iram Mahmood Chunzhao Liu Huizhou Liu 《Journal of Molecular Catalysis .B, Enzymatic》2009,58(1-4):103-109
Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C1, C4 and C8) and anions (Cl−, BF4− and PF6−). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme. 相似文献
12.
The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO32−35 mM and at an ionic strength of 0.1 M (LiClO4). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M−1 s−1 with ΔH≠ = (13.2±0.7) kcal mol−1 and ΔS≠ = (−15.1 ± 0.7) cal deg−1 mol−1. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes. 相似文献
13.
The thermal coefficient of expansion of egg lecithin bilayer thickness, αd1, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd1 changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd1 decreases monotonically from −2·10−3 for pure egg lecithin to −1·10–3 at mole ratio 1:1. Below 12 °C αd1 is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10−3 at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium. 相似文献
14.
Emission of N2O, N2, CH4, and CO2 from constructed wetlands for wastewater treatment and from riparian buffer zones 总被引:3,自引:1,他引:2
We measured nitrous oxide (N2O), dinitrogen (N2), methane (CH4), and carbon dioxide (CO2) fluxes in horizontal and vertical flow constructed wetlands (CW) and in a riparian alder stand in southern Estonia using the closed chamber method in the period from October 2001 to November 2003. The replicates’ average values of N2O, N2, CH4 and CO2 fluxes from the riparian gray alder stand varied from −0.4 to 58 μg N2O-N m−2 h−1, 0.02–17.4 mg N2-N m−2 h−1, 0.1–265 μg CH4-C m−2 h−1 and 55–61 mg CO2-C m−2 h−1, respectively. In horizontal subsurface flow (HSSF) beds of CWs, the average N2 emission varied from 0.17 to 130 and from 0.33 to 119 mg N2-N m−2 h−1 in the vertical subsurface flow (VSSF) beds. The average N2O-N emission from the microsites above the inflow pipes of the HSSF CWs was 6.4–31 μg N2O-N m−2 h−1, whereas the outflow microsites emitted 2.4–8 μg N2O-N m−2 h−1. In VSSF beds, the same value was 35.6–44.7 μg N2O-N m−2 h−1. The average CH4 emission from the inflow and outflow microsites in the HSSF CWs differed significantly, ranging from 640 to 9715 and from 30 to 770 μg CH4-C m−2 h−1, respectively. The average CO2 emission was somewhat higher in VSSF beds (140–291 mg CO2-C m−2 h−1) and at the inflow microsites of HSSF beds (61–140 mg CO2-C m−2 h−1). The global warming potential (GWP) from N2O and CH4 was comparatively high in both types of CWs (4.8 ± 9.8 and 6.8 ± 16.2 t CO2 eq ha−1 a−1 in the HSSF CW 6.5 ± 13.0 and 5.3 ± 24.7 t CO2 eq ha−1 a−1 in the hybrid CW, respectively). The GWP of the riparian alder forest from both N2O and CH4 was relatively low (0.4 ± 1.0 and 0.1 ± 0.30 t CO2 eq ha−1 a−1, respectively), whereas the CO2-C flux was remarkable (3.5 ± 3.7 t ha−1 a−1). The global influence of CWs is not significant. Even if all global domestic wastewater were treated by wetlands, their share of the trace gas emission budget would be less than 1%. 相似文献
15.
A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The Km and kcat values for the pure enzyme were 1.35 mg ml−1 and 249 μM mg−1 min−1, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l−1 gelatinized soluble starch or 125 g l−1 raw wheat starch at an enzyme concentration of 10 U g−1 of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively. 相似文献
16.
Gerhard Fleischmann Miguel Beato 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,540(3):500-517
The synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione (Org 2058) was used to characterize the progesterone receptor in the uterine cytosol of the rabbit. [3H] Org 2058 binds to a homogeneous population of protin binding sites with an apparent association equilibrium constant of 7.7· 108 M−1 at 0°C. The concentration of protein-bound steroid at saturation is 2.3 pmol per mg of cytosol protein. [3H] Progesterone binds to the same set of binding sites but exhibits a 4–5 fold lower apparent association constant. The difference in affinity is mainly due to a 13-fold slower rate of dissociation of the synthetic progestin compared with progesterone. Org 2058 competes very efficiently for the binding of [3H] progesterone to the uterine cytosol, and progesterone also competes, although less efficiently, for the binding of [3H]-Org 2058. There is a good correlation between the progestational activity of various steroids and their ability to compete with [3H] Org 2058 binding to the cytosol. At 0°C, there is no metabolic transformation of either Org 2058 or progesterone in the uterine cytosol.When filled with the steroid, the progesterone receptor is stable, but in the absence of the steroid the receptor binding sites are thermolabile and show a rapid decay at 20°C
. Org 2058 is more effective than progesterone in protecting the receptor against thermal inactivation. The rate constant of association and dissociation of [3H] Org 2058 and the cytosol receptor are strongly dependent on temperature and the activation energy of the dissociation reaction is 17.8 kcal/mol. The equilibrium association constant is less dependent on temperature and exhibits ΔH° of −4.7 kcal/mol. The binding reaction shows a positive entropy change of 23 cal · K−1 · mol−1.At low ionic strength the complex of Org 2058 and the progesterone receptor tends ot aggregate. It sediments as a broad peak on sucrose gradients (4–6 S), and is excluded from columns of Sephadex G-100 and G-200. At concentrations of NaCl above 0.15 M, the receptor sediments in sucrose gradients as an homogeneous peak at 3.6 S, but upon gel filtration it aggregates and a complex elution pattern is observed, that prevents a precise estimation of the molecular weight. 相似文献
17.
Randall K Packer Jeffrey L Garvin 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,120(4):777-783
We measured Na+/K+ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na+/K+ ATPase activity was measured at 8, 17, 25, and 37°C. Vmax values for winter fish increased from 0.48±0.07 μmol P mg−1 protein h−1 at 8°C to 7.21±0.79 μmol P mg−1 protein h−1 at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg−1 protein h−1. The Km for ATP and for Na+ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na+/K+ ATPase from summer fish was 10 309 (μmol P mg−1 h−1) K−1. In winter fish, the Km for ATP and Na+ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The Km values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression. 相似文献
18.
P. Campíns-Falc C. Molins-Legua A. Sevillano-Cabeza L. A. Tortajada Genaro 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,759(2):1162
A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10−2 min−1 in solution versus 7.7±1.1×10−4 min−1 on the (C18) solid support. The results obtained showed that forming the derivative on the packing support (C18) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples. 相似文献
19.
Ruri Kikura Yuji Nakahara Shigeo Kojima 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2000,741(2):3139
In order to study the disposition of dimethylamphetamine (DMAP) and its metabolites, DMAP N-oxide, methamphetamine (MA) and amphetamine (AP), from plasma to hair in rats, a simultaneous determination method for these compounds in biological samples using gas chromatography–mass spectrometry with selected ion monitoring (GC–MS-SIM) was developed. As DMAP N-oxide partially degrades to DMAP and MA during GC–MS analysis, it was necessary to avoid conditions which co-extract the N-oxide in the sample preparation so as to assure no contribution of artifactual products from DMAP N-oxide in the detection of the other compounds. For confirmation of the satisfactory separation of DMAP N-oxide from the others, the internal standards used for quantification were labeled with different numbers of deuterium atoms. Determination of unchanged DMAP was performed without any derivatization, that of DMAP N-oxide was carried out after conversion into trifluoroacetyl-MA by reaction with trifluoroacetic anhydride, and MA and AP were quantified after trifluoroacetyl-derivatization.After intraperitoneal administration of DMAP HCl to pigmented hairy rats (5 mg kg−1 day−1, 10 days, n=3), concentrations of DMAP and its metabolites in urine, plasma and hair were measured by GC–MS-SIM. The area under the concentration versus time curves (AUCs) of DMAP, DMAP N-oxide, MA and AP in the plasma were 397.2±97.5, 279.7±68.3, 18.4±1.2 and 15.9±2.2 μg min ml−1, while their concentrations in the hair newly grown for 4 weeks after administration were 4.82±0.67. 0.45±0.09, 3.25±0.36 and 0.89±0.05 ng mg−1, respectively. This fact suggested that the incorporation tendency of DMAP N-oxide from plasma into hair was distinctly low in comparison with the other compounds. 相似文献
20.
An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH4)2SO4 precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, Km 0.29 mM, Vmax 2.56 μkat mg−1) and pNP-β-d-GalNAc (optimum pH 4.4, Km 0.27 mM, Vmax 2.50 μkat mg−1). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The Kis for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)2 and pNP-β-d-(GlcNAc)3 than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)2 (optimum pH 5.0) and (GlcNAc)3−6 (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)n were predominantly (GlcNAc)n−1 and GlcNAc. 相似文献