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1.
6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q10 and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium Random Bi Bi” kinetic model with Km values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/Vm versus 1/6PG and 1/NADP plots gave a Vm value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a Ki of 31.91±1.31 μM. The relatively small Ki for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.  相似文献   

2.
The Ca2+-activated maxi K+ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca2+-permeable cation channels, because the maxi K+ channel was not stretch-activated in both the cell-attached configuration using Ca2+-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K+ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K+ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K+ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV t ) when luminal K+ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV t =−7.2 ± 0.5 mV in control vs.ΔV t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca2+-activated maxi K+ channel is responsible for flow-dependent K+ secretion by coupling with the stretch-activated Ca2+-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998  相似文献   

3.
The hypothesis was tested that invasive trees have hydraulic traits that contribute to their invasive nature. Five pairs of co-occurring invasive and native trees, in mesic habitats, were selected: (1) Tamarix ramosissima and Salix amygdaloides; (2) Robinia pseudoacacia and Alnus rhombifolia (3) Schinus terebinthifolius and Myrica cerifera; (4) Ligustrum sinense and Acer negundo; and (5) Sapium sebiferum and Diospyros virginiana, respectively. Resistance to cavitation (the water potential [Ψ x ] at 75% loss of hydraulic conductivity [Ψ75]) was not consistently greater for invasive compared to native species (Ψ75=−1.91 and −1.67 MPa, respectively). Xylem specific conductivity (K s), a measure of xylem efficiency, was not different between native and invasive species (K s = 3.50 and 3.70 kg s−1 MPa−1 m−1, respectively). The lack of difference for resistance to cavitation among invasive and native species suggests that the sampled invaders are not more tolerant to water stress than co-occurring native species. Apparently the spread and invasive nature of the sampled species cannot be explained by hydraulic traits alone.  相似文献   

4.
Purified uricase from a caprine kidney, possessed K m and V max values of 1.1 mg ml−1 and 3512 IU (mg protein)−1 for uric acid hydrolysis, respectively. The optimum temperature and pH for catalytic activity were 40 °C and 8.5, respectively. The activation energy for formation of ES complex was 13.6 kJ mol−1. Enthalpy (ΔH*), entropy of activation (ΔS*) and Gibbs free energy demand of uricase inactivation were 62.8 kJ mol−1, −102 J mol−1 K−1 and 104.3 kJ mol−1, respectively. Gibbs free enrgy demand for substrate binding and transition state stabilization were also determined which were comparable with those for themostable enzymes.  相似文献   

5.
The responses of freshly isolated hippocampal pyramidal neurons to rapid, elevations of the external potassium concentration ([K+] out ) were investigated using the whole-cell variation of a patch-clamp technique. An elevation of [K+] out induced a two-phase inward current at the membrane potentials more negative than the reversal potential for K ions. This current consisted of a leakage, current and a time-dependent current (τ=40–50 msec at 21°C), the latter designated below asI ΔK. It displayed first-order activation kinetics that showed neither voltage, nor concentration dependence. The amplitude of this current was determined by the external K+ concentration and increased with hyperpolarization. Voltage dependence ofI ΔK measured within the range from −20 to −120 mV was similar to that for inward rectifier. Activation ofI ΔK was utterly dependent on Na+; substitution of extracellular Na+ with choline chloride almost completely depressedI ΔK.I ΔK was absent in the cells freshly dissociated from the nodosal and dorsal root ganglia. This suggests that this earlier unrecognized current is instrumental in preserving densely packed hippocampal pyramidal neurons from sudden increases in [K+] out and following spontaneous over-excitation. It prevents the neurons from responding to K+-induced depolarizations by slowing down potassium influx.  相似文献   

6.
Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.  相似文献   

7.
Xylanases produced from a locally isolated strain of Thermomyces lanuginosus and its mutant derivative were purified to a yield of 39.1 and 42.83% with specific activities of 15,501 and 17,778 IU mg−1 protein, respectively. The purification consisted of two steps i.e., ammonium sulphate precipitation, and gel filtration chromatography. The mutant enzyme showed high affinity for substrate, with a K m of 0.098 mg ml−1 as compared to wild type enzyme showing K m of not less than 0.112 mg ml−1. It was found that pH values of 8.1 and 7.3 were best for activity of the mutant and wild-type-derived enzymes, respectively. The values of pK a of the acidic limbs of both enzymes were the same (5.0 and 4.9, respectively) but the pK a value of the basic limb was slightly increased, indicating the participation of a carboxyl group present in a non-polar environment. Temperatures of 70 and 65°C were found optimal for mutant and wild-derived xylanase, respectively. Enzymes displayed a high thermostability showing a half life of 31.79 and 6.0 min (5.3-fold improvement), enthalpy of denaturation (ΔH*) of 146.06 and 166.95 kJ mol−1, entropy of denaturation (ΔS*) of 101.44 and 174.67, and free energy of denaturation (ΔG*) of 110.25 and 105.29 kJ mol−1 for mutant- and wild-organism derived enzyme, respectively at 80°C. Studies on the folding and stability of cellulase-less xylanases are important, since their biotechnological employments require them to function under extreme conditions of pH and temperature. The kinetic and thermodynamic properties suggested that the xylanase from the mutant organism is better as compared to xylanase produced from the wild type and previously reported strains of same species, and may have a potential usage in various industrial fields.  相似文献   

8.
Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·106 M−1K≤1.4·106 M−1, free energy ΔG o=−8.7±0.3 kcal/mol, enthalpy ΔH o≅0, and entropy ΔS o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10−5 M EtBr) increased by 17°C as compared with the ΔT m of free homopolymer, whereas the half-width of the transition (T m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K 1=1.7·105 M−1; ΔG o=−8.10±0.03 kcal/mol) and weak (K 2=2.9·103 M−1; ΔG o=−6.0±0.3 kcal/mol).The ΔG o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.  相似文献   

9.
Elevation of the external potassium concentration induced a two-phase inward current in freshly isolated pyramidal hippocampal neurons. This current was voltage-dependent and demonstrated strong inward rectification. The current consisted of a leakage current and a time-dependent current (τ=40–50 msec at 21°C); the latter was designated asI ΔK. As was shown earlier, K+ is a major charge carrier in the development of slow potassium-activated current. The pharmacological properties ofI ΔK were studied using a patch-clamp technique.I ΔK was completely blocked by external 10 mM TEA or 5 mM Ba2+ (IC50=480±90mM) and exhibited low sensitivity to extracellular Cs+ (2 mM). This current was not affected by 1 mM 4-aminopyridine and was insensitive to a muscarinic agonist, carbachol (50 μM), and to 1 mM extracellular Cd2+. Elevation of external Ca2+ from 2.5 mM to 10 mM did not changeI ΔK. Our data indicate that the pharmacological properties ofI ΔK differ from those of other voltage-gated potassium currents, but more specific blockers must be used to make this evidence conclusive.  相似文献   

10.
A. niger produced α-glucosidase, α-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5–9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65°C, respectively, and were stable for 1 h at temperatures of up to 60°C. The kinetic parametersK m andV showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.  相似文献   

11.
Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg2+and Br ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn2+, Cu2+, K+, Cd2+, Ag+, Fe3+, Mn2+, Co2+, Hg2+, Pb2+ and Sn2+), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E a of these reactions and temperature dependence (at 0–30°C) of k cat, k cat/K m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k cat and k cat/K m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.  相似文献   

12.
A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase (frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter. The resulting strain SZ420 (ΔfrdBC ΔldhA ΔackA ΔfocA-pflB ΔpdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90% under anaerobic conditions.  相似文献   

13.
Characean internodal cells generate receptor potential (ΔE m) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated ΔE m at the moment of both compression and decompression, and the amplitude of ΔE m at the moment of decompression, (ΔE m)E, was larger than that at compression. The long-lasting stimulus caused a membrane deformation (ΔD m) having two components, a rapid one, (ΔD m)rapid, at the moment of compression and a slower one, (ΔD m)slow, during the long-lasting compression. We assumed that (ΔD m)slow might have some causal relation with the larger ΔE m at (ΔE m)E. We treated internodal cells with either HgCl2 or ZnCl2, water channel inhibitors, to decrease (ΔD m)slow. Both inhibitors attenuated (ΔD m)slow during compression. Cells treated with HgCl2 generated smaller (ΔE m)E compared to nontreated cells. On the other hand, cells treated with ZnCl2 never attenuated (ΔE m)E but, rather, amplified it. Thus, the amplitude of (ΔD m)slow did not always show tight correlation with the amplitude of (ΔE m)E. Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (ΔE m)E. Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca2+ channel.  相似文献   

14.
Increasing leaf photosynthesis offers a possible way to improve yield potential in rice (Oryza sativa L.). Carbon isotope discrimination (Δ13C) has potential as an indirect selection criterion. In this study, we searched for quantitative trait loci (QTLs) controlling Δ13C, and assessed their association with leaf photosynthesis. Substitution mapping by using chromosome segment substitution lines (CSSLs), that carry segments from the indica cultivar Kasalath in the genetic background of the japonica cultivar Koshihikari, identified genomic regions affecting Δ13C on chromosomes (Chr.) 2, 3, 6, 7, and 12. One of the CSSLs, SL208, in which most regions on Chr. 3 were substituted with Kasalath segments, showed higher leaf stomatal conductance for CO2 (g s) and Δ13C than Koshihikari during the vegetative stage although leaf photosynthetic rate did not differ between them. These results suggest an association between Δ13C and g s. To test this association, we performed a QTL analysis for Δ13C at vegetative and heading stages in an F2 population derived from a cross between SL208 and Koshihikari. The results confirmed a QTL controlling Δ13C on the long arm of Chr. 3. By using a near-isogenic line specific to Hd6, we ruled out the possibility that variation in Δ13C was generated through the pleiotropic effect of heading date.  相似文献   

15.
In a multiple deletion mutanthxt1Δhxt2Δhxt3Δ hxt4Δsnf3Δ ofSaccharomyces cerevisiae growing on 2 % glucose, high-affinity glucose-uptake (lowK m) was exhibited throughout growth on glucose in contrast to the wild-type, which exhibited the usual low-affinity to high-affinity transition as the glucose in the medium was consumed. elevated levels of invertase activity throughout growth on glucose, in this mutant as compared to the wild-type, indicate that glucose repression may be impaired. Howver, in a mutant containing only theHXT2 gene (hxt1Δhxt3Δhxt4Δ snf3Δ), invertase levels were similar to those in the wild-type. It is likely, therefore, that some of these putative glucose transporters, such asHXT2, also have regulatory roles in cellular metabolism. In triple hexose-kinase mutants, rapid (200-ms) measurements of initial glucose-uptake revealed high-affinity glucose uptake (K m approx. 2 mmol/L) while measurements on the slower 5-s scale clearly demonstrate that uptake is not linear over this longer period. These results suggest that this high-affinity component does not require a functional hexose-kinase.  相似文献   

16.
Two respiratory-deficient nuclear petites, FY23Δpet191 and FY23Δcox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23ρ0. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K i, of 2.3% (w/v) and a specific rate of ethanol production, q p, of 0.90 g ethanol g dry cells−1 h−1. FY23ρ0 was the most sensitive to ethanol, exhibiting a K i of 1.71% (w/v) and q p of 0.87 g ethanol g dry cells−1 h−1. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23Δpet191, having a K i of 2.14% (w/v) and the 85% respiratory-deficient FY23Δcox5a, having a K i of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23ρ0 is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation. Received: 22 September 1997 / Accepted: 7 December 1997  相似文献   

17.
It has recently been reported that N2 fixation and carbon isotope discrimination (Δ) are negatively correlated. To further test this hypothesis, a greenhouse experiment was conducted to investigate if Δ is correlated with the efficiency of lentil (Lens culinaris cv Laird) in fixing atmospheric nitrogen. Lentil seed was inoculated with one of 10 Rhizobium leguminosarum strains that varied in their effectiveness in symbiotic N2 fixation. Carbon-13 discrimination was positively correlated with N2 fixation (r2=0.60*). Although the amount of N2 fixed ranged from 1.5 mg N to 13.5 mg N shoot−1, the range of Δ values was only 25.8 to 26.6%.. It is unlikely that variability of such small magnitude could be of any practical use in selecting for N2-fixing efficiency.  相似文献   

18.
19.
SPINDLY (SPY) is an important regulator of plant development, and consists of an N-half tetratricopeptide repeat (TPR) domain containing 10 TPR motifs and a C-half catalytic domain, similar to O-GlcNAc transferase (OGT) of animals. The best characterised role of SPY is a negative regulator of GA signalling, and all known spy alleles have been isolated based on increased GA response. Of the eight alleles that directly affect the TPR domain, all alter TPRs 6, 8 and/or 9. To test the hypothesis that a subset of TPRs, including 6, 8 and 9, are both essential and sufficient for the regulation of GA response, we overexpressed the full-length barley (Hordeum vulgare L.) SPY protein (HvSPY) and several deletion mutants in barley aleurone cells and in Arabidopsis wild type (WT) and spy-4 plants. Transient assays in barley aleurone cells, that also express endogenous HvSPY, demonstrated that introduced HvSPY and HvTPR inhibited GA3-induced α-amylase expression. With the exception of HvSPYΔ1–5, the other deletion proteins were partially active in the barley assay, including HvSPYΔ6–9 which lacks TPRs 6, 8 and 9. In Arabidopsis, analysis of seed germination under a range of conditions revealed that 35S:HvSPY increased seed dormancy. Hvspy-2, which lacks parts of the eighth and ninth TPRs, was able to partially complement all aspects of the spy-4 phenotype. In the presence of AtSPY, 35S:HvTPR caused some phenotypes consistent with a decrease in GA signalling, including increased seed sensitivity to paclobutrazol and delayed flowering. These plants also possessed distorted leaf morphology and altered epidermal cell shape. Thus, despite genetic analysis demonstrating that TPRs 6, 8 and 9 are required for regulation of GA signalling, our results suggest that these TPRs are neither absolutely essential nor sufficient for SPY activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

20.
Spreading evidence suggests that geomagnetic field (GMF) modulates artificial magnetic fields biological effect and associated with increased cardiovascular morbidity. To explore the underlying physiological mechanism we studied 350 mT static magnetic field (SMF) effect on arterial baroreflex-mediated skin microcirculatory response in conjunction with actual geomagnetic activity, reflected by K and K p indices. Fourteen experiments were performed in rabbits sedated by pentobarbital infusion (5 mg/kg/h). Mean femoral artery blood pressure, heart rate, and the ear lobe skin microcirculatory blood flow, measured by microphotoelectric plethysmogram (MPPG), were simultaneously recorded before and after 40 min of NdFeB magnets local exposure to sinocarotid baroreceptors. Arterial baroreflex sensitivity (BRS) was estimated from heart rate/blood pressure response to intravenous bolus injections of nitroprusside and phenylephrine. We found a significant positive correlation between SMF-induced increase in BRS and increment in microvascular blood flow (ΔBRS with ΔMPPG, r=0.7, p<0.009) indicated the participation of the arterial baroreflex in the regulation of the microcirculation and its enhancement after SMF exposure. Geomagnetic disturbance, as opposed to SMF, decreased both microcirculation and BRS, and counteracted SMF-induced increment in microcirculatory blood flow (K-index with ΔMPPG; r s=−0.55, p<0.041). GMF probably affected central baroreflex pathways, diminishing SMF direct stimulatory effect on sinocarotid baroreceptors and on baroreflex-mediated vasodilatatory response. The results herein may thus point to arterial baroreflex as a possible physiological mechanism for magnetic-field cardiovascular effect. It seems that geomagnetic disturbance modifies artificial magnetic fields biological effect and should be taken into consideration in the assessment of the final effect. An erratum to this article can be found at  相似文献   

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