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1.
The analysis of kinetic and thermodynamic parameters of binding of peptide and nonpeptide dimerization inhibitors of HIV protease (HIVp) to the enzyme monomers immobilized on an optical chip has been studied by surface plasmon resonance. The molecular interactions were investigated at different inhibitor concentrations (0–80 μM) and temperatures (15–35°C). Determination of kinetic (k on, k off), equilibrium (K d), and thermodynamic (ΔG, ΔH, and -TΔS) has shown that both inhibitors are characterized by similar interaction parameters and the entropic term (-TΔS) of about −20 kcal/mol is the main driving force for the HIVp complex formation with the inhibitors, while the positive value (14 kcal/mol) of the enthalpic term (ΔH) counteracted the complex formation.  相似文献   

2.
Two approaches to determine the fraction (μ) of mitochondrial respiration sustained during illumination by measuring CO2 gas exchange are compared. In single leaves, the respiration rate in the light (`day respiration' rate Rd) is determined as the ordinate of the intersection point of A–ci curves at various photon flux densities and compared with the CO2 evolution rate in darkness (`night respiration' rate Rn). Alternatively, using leaves with varying values of CO2 compensation concentration (Γ), intracellular resistance (ri) and Rn, an average number for μ can be derived from the linear regression between Γ and the product riċRn. Both methods also result in a number c* for that intercellular CO2 concentration at which net CO2 uptake rate is equal to –Rd. c* is an approximate value of the photocompensation point Γ* (Γ in the absence of mitochondrial respiration), which is related to the CO2/O2 specificity factor of Rubisco Sc/o. The presuppositions and limitations for application of both approaches are discussed. In leaves of Nicotiana tabacum, at 22 °C, single leaf measurements resulted in mean values of μ = 0.71 and c* = 34 μmol mol−1. At the photosynthetically active photon flux density of 960 μmol quanta m−2 s−1, nearly the same numbers were derived from the linear relationship between Γ and riċRn. c* and Rd determined by single leaf measurements varied between 31 and 41 μmol mol−1 and between 0.37 and 1.22 μmol m−2 s−1, respectively. A highly significant negative correlation between c* and Rd was found. From the regression equation we obtained estimates for Γ* (39 μmol mol−1), Sc/o (96.5 mol mol−1) and the mesophyll CO2 transfer resistance (7.0 mol−1 m2 s). This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

3.
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   

4.
Antibody-directed enzyme prodrug therapy (ADEPT) may improve the therapeutic index of cytostatic agents. We compared two prodrugs, epirubicin-glucuronide (Epi-glu) and doxorubicin-spacer-glucuronide (Dox-sp-glu), for their cytotoxicity on activation by a monoclonal antibody-enzyme conjugate bound to tumor cells. The results showed that the prodrugs were 10 (Dox-sp-glu) and 100 (Epi-glu) times less toxic than the parent drugs against OVCAR-3 cells. This difference was a result of the hydrophilic property of the prodrugs resulting in a reduced cellular uptake. The enzyme-catalyzed hydrolysis of Dox-sp-glu byE. coli-derived β-glucuronidase (GUS) (K m 500 μM,V max 21,000 μmol/min/g) was much more efficient than that of Epi-glu (K m 10 μM,V max 40 μmol/min/g). Incubation of OVCAR-3 cells with an enzyme-immunoconjugate prepared from monoclonal antibody 323/A3 andE. coli-derived GUS before treatment with prodrugs completely restored the cytotoxicity of the prodrugs to the level of the parent drugs.  相似文献   

5.
Cultures able to dechlorinate cis-1,2-dichloroethene (cDCE) were selected with ethene (3–20%, v/v) as the sole source of carbon and energy. One mixed culture (K20) could degrade cDCE (400 μmol l–1) or vinyl chloride (100 μmol l–1) in the presence of ethene (≤ 80 μmol l–1 and ≤ 210 μmol l–1, respectively). This culture consists of at least five bacterial strains. All five strains were able to degrade cDCE cometabolically in pure culture. The mixed culture K20 was highly tolerant against cDCE (up to 6 mmol l–1 in the liquid phase). Degradation of cDCE (200 μmol l–1) was not affected by the presence of trichloroethene (100 μmol l–1) or tetrachloroethene (100 μmol l–1). Transformation yields (Ty, defined as unit mass of chloroethene degraded per unit mass of ethene consumed) of the mixed culture K20 were relatively high (0.51 and 0.61 for cDCE and vinyl chloride, respectively). The yield for cDCE with ethene as auxiliary substrate was ninefold higher than any values reported with methane or methane/formate as auxiliary substrate. The viability of the cells of the mixed culture K20 (0.3 mg of cells ml–1) was unaffected by the transformation of ≤ 200 μmol l–1 cDCE in 300 min. Received: 9 March 1999 / Accepted: 21 July 1999  相似文献   

6.
In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604–610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of α-methyl-d-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K i for phlorizin increased at least by a factor of 5 compared to the wild-type K i of 4.6 ± 0.7 μmol/l; most striking changes were observed for Y604K (K i = 75.3 ± 19.0 μmol/l) and C608K (K i = 83.6 ± 13.9 μmol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (K m = 0.8 ± 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside. Received: 29 March 2001/Revised: 15 June 2001  相似文献   

7.
Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m7Guo), and orthophosphate (Pi, natural cosubstrate) and its thiophosphate analogue (SPi), found to be a weak cosubstrate, have been studied in the pH range 5–8. In this pH range Guo and Ino exist predominantly in the neutral forms (pKa 9.2 and 8.8); m7Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pKa 7.0); and Pi and SPi exhibit equilibria between monoanionic and dianionic forms (pKa 6.7 and 5.4, respectively). The phosphorolysis of m7Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SPi, independently of pH. With Pi, the human enzyme shows Michaelis kinetics only at pH ∼5. However, in the pH range 5–8 for the bacterial enzyme, and 6–8 for the human enzyme, enzyme kinetics with Pi are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by Pi, characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K m1 and K m2) and apparent maximal velocities (V max1 and V max2). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K m1 < K m2) and enzyme kinetics (V max1/K m1 > V max2/K m2). Comparison of the pH-dependence of the substrate properties of Pi versus SPi points to both monoanionic and dianionic forms of Pi as substrates, with a marked preference for the dianionic species in the pH range 5–8, where the population of the Pi dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n H ∼1 to n H ∼0.7 for Guo with the human enzyme, and to n H ∼0.7 and 0.5 for m7Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SPi in relation to its structure.  相似文献   

8.
The study showed that the genetically engineered microorganism (GEM) bioaugment successfully the dye wastewater biotreatment systems to enhance acid red GR (ARGR) removal. Escherichia coli JM109 (pGEX-AZR) was the GEM with higher azoreductase activity. The kinetics of the ARGR decolorization by the E. coli JM109 (pGEX-AZR) agreed with Andrews model. The kinetic parameters, r dye,max, K s and K i , were found to be 42.45 mg g−1 h−1, 584.93 mg L−1 and 556.89 mg L−1, respectively. The E. coli JM109 (pGEX-AZR) was tested in anaerobic sequencing batch reactors (AnSBR) in order to enhance the ARGR decolorization. The decolorization rate of ARGR was affected by the amount of E. coli JM109 (pGEX-AZR) inoculation and the best amount of inoculation was 10%. The continuous operations of the four bioreactors with different E. coli JM109 (pGEX-AZR) immobilization supports showed that the E. coli JM109 (pGEX-AZR) could bioaugment decolorization in AnSBRs with suspended and immobilized on macroporous foam carriers. For 42 days continuous operation in the AnSBRs, both the tolerance to ARGR concentration shock and the decolorization rate in these two bioaugmented AnSBRs are higher than those of the other two systems, control system and bioaugmented AnSBRs system with the sodium-alginate immobilized cells, the decolorization rate reached 90%. Changes in microbial community were detected by ribosomal intergenic spacer analysis (RISA) and amplified ribosomal DNA restriction analysis (ARDRA), which revealed that the introduced E. coli JM109 (pGEX-AZR) was persistent in the augmented systems and maintained higher metabolic activity.  相似文献   

9.
Simultaneous curve fitting for the ionization parameters of oxidized and reduced horse heart cytochrome c in 0.15M KCl and 20°C yields values for the ionization constants (as pK′) and the heats of ionization (ΔHi) which can reconstruct either the potentiometric or thermal titration curves. Reduced cytochrome c requires 8 sets of groups, whereas oxidized cytochrome c requires 10 sets of groups. The additional groups in the oxidized preparation appear to involve the ferriheme (pK′, 9.25; ΔHi, 13.7 kcal/mol) and a tyrosine (pK′ ? 10.24) that is not present in the reduced form. The potentiometric and thermal difference curves (reduced – oxidized) involve the appearance of 17 kcal/mol centered at pH 9.7 and 5.8 kcal/mol centered at pH 4.9. The carboxyl groups in both species appear to be normal for the hydrogen-bonded form. Only one histidine has normal ionization properties (pK′, 6.7; ΔHi, 7.5 kcal/mol), as do 17 of the lysine residues (pK′, 10.8; ΔHi, 11.5 kcal/mol).  相似文献   

10.
The transformation capacity (Tc) ofMethylosinus trichosporium OB3b in the degradation of ethylene chlorides was determined by measuring the decrease of soluble methane monooxygenase (sMMO) activity of resting cells in batch experiments. All measurements of sMMO activity were taken in the presence of 20 mM formate to avoid the deficiency of reducing power, and within 2 hrs to avoid the effect of natural inactivation from instability of the resting cells. The constant Tc values of 0.58±0.132 and 0.80±0.17 μmol/mg cell were obtained for trichloroethylene (TCE) and 1,2-dichloroethylene (cis andtrans-1,2-DCE), respectively, regardless of their concentrations. The transformation capacity measured by this method can be used to predict the amount of cells that should be stimulated inin-situ bioremediation.  相似文献   

11.
Salt-tolerant aromatic yeast is an important microorganism arising from the solid state fermentation of soy sauce. The fermentation kinetics of volatile esters by Candida etchellsii was studied in a batch system. The data obtained from the fermentation were used for determining the kinetic parameters of the model. Batch experimental results at four NaCl levels (180, 200, 220, and 240 g/L) were used to formulate the parameter estimation model. The kinetic parameters of the model were optimized by specifically designed Runge-Kutta Genetic Algorithms (GA). The resulting mathematical model for volatile ester production, cell growth and glucose consumption simulates the experimental data well. The resulting new model was capable of explaining the behavior of volatile ester fermentation. The optimized parameters (μo, X max, K i, α, β, Y X/S, m, and Y P/S) were characterized by a correlation of functions assuming salinity dependence. The kinetic models optimized by GA describe the batch fermentation process adequately, as demonstrated by our experimental results.  相似文献   

12.
The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin, was studied in the methemalbumin-H2O2-tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in a buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K i, μM) and the inhibition degree (%). The K i values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 μM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity of esculetin in peroxidase-catalyzed TMB oxidation at pH 6.4 is characterized by a K i value equal to 1.15 μM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant of plant origin among all derivatives previously studied in model biochemical systems.  相似文献   

13.
The lipid bilayer technique was used to examine the effects of the ATP-sensitive K+ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I max and average mean current, I′) and kinetic parameters (frequency of opening F o , probability of the channel being open P o and mean open time, T o , of the SCl channel. Glibenclamide increased mean closed time, T c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P o , and T o in the absence of ATP and Mg2+ but it had no effect on I max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I max, whereas cromakalim reduced P o and I′ by increasing channel transitions to the closed state and reducing T o without affecting I max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] cis or [glibenclamide] cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] cis and [glibenclamide] cis highlights the therapeutic potential of these drugs in regulating the Ca2+-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998  相似文献   

14.
A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 μg/l. This resulted in the calculation of the following kinetic parameters (μmax = 0.19 h−1, K s = 2.9 μg/l, K i = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE. Received: 16 July 1998 / Received revision: 11 November 1998 / Accepted 31 November 1998  相似文献   

15.
The oxidation and growth kinetics of ferrous iron with Thiobacillus ferrooxidans in continuous cultures was examined at several total iron concentrations. On-line off-gas analyses of O2 and CO2 were used to measure the oxygen and carbon dioxide consumption rates in the culture. Off-line respiration measurements in a biological oxygen monitor (BOM) were used to measure directly the maximum specific oxygen consumption rate, qO2,max, of cells grown in continuous culture. It was shown that these reproducibly measured values of qO2,max vary with the dilution rate. The biomass-specific oxygen consumption rate, qO2, is dependent on the ratio of the ferric and ferrous iron concentrations in the culture. The oxidation kinetics was accurately described with a rate equation for competitive ferric iron inhibition, using the value of qO2,max measured in the BOM. Accordingly, only the kinetic constant Ks/K i needed to be fitted from the measurements. A new method was introduced to determine the steady-state kinetics of a cell suspension in a batch culture that only takes a few hours. The batch culture was set up by terminating the feeding of a continuous culture at its steady state. The kinetic constant K s/K i determined in this batch culture agreed with the value determined in continuous cultures at various steady states. Received: 8 February 1999 / Accepted: 17 February 1999  相似文献   

16.
Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K m of 0.16 mM and a V max of 81 μmol of trehalose (min)−1 (mg of protein)−1 at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.  相似文献   

17.
Sah S  Phale PS 《Biodegradation》2011,22(3):517-526
1-Naphthol 2-hydroxylase (1-NH) which catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene was purified to homogeneity from carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a homodimer with subunit molecular weight of 66 kDa. UV, visible and fluorescence spectral properties, identification of flavin moiety by HPLC as FAD, and reconstitution of apoenzyme by FAD suggest that enzyme is FAD-dependent. 1-NH accepts electron from NADH as well as NADPH. Besides 1-naphthol (K m, 9.1 μM), the enzyme also accepts 5-amino 1-naphthol (K m, 6.4 μM) and 4-chloro 1-naphthol (K m, 2.3 μM) as substrates. Enzyme showed substrate inhibition phenomenon at high concentration of 1-naphthol (K i, 283 μM). Stoichiometric consumption of oxygen and NADH, and biochemical properties suggest that 1-NH belongs to FAD containing external flavomonooxygenase group of oxido-reductase class of enzymes. Based on biochemical and kinetic properties, 1-NH from Pseudomonas sp. strain C6 appears to be different than that reported earlier from Pseudomonas sp. strain C4. Chemical modification and protection by 1-naphthol and NADH suggest that His, Arg, Cys, Tyr and Trp are at or near the active site of 1-NH.  相似文献   

18.
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   

19.
The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate (Pi). Apparent K m values for ADP and Pi were 7–11 and 60–120 μM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg2+). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly: K m value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent K m values for ADP and Pi (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006) Biochemistry, 45, 14552–14558).  相似文献   

20.
The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 ± 1.6 μM, Ki = 38 ± 1.4 μM) and delF508 CFTR (Ks = 3 ± 1.8 μM, Ki = 33 ± 1 μM). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 ± 1.13 μM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 ± 1.01 μM) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.  相似文献   

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