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1.
The acid-base properties of Adenosine 5'-triphosphate (ATP) in NaCl and KCl aqueous solutions at different ionic strengths (0相似文献
2.
The acid-base properties of γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) were determined by potentiometry (ISE-H(+), glass electrode) in pure NaI((aq)) and in NaCl((aq))/MgCl(2(aq)), and NaCl((aq))/CaCl(2(aq)) mixtures, at T = 298.15 K and different ionic strengths (up to I(c) ~ 5.0 mol L(-1)). In addition, the activity coefficients of glutathione were also determined by the distribution method at the same temperature in various ionic media (LiCl((aq)), NaCl((aq)), KCl((aq)), CsCl((aq)), MgCl(2(aq)), CaCl(2(aq)), NaI((aq))). The results obtained were also used to calculate the Specific ion Interaction Theory (SIT) and Pitzer coefficients for the dependence on medium and ionic strength of glutathione species, as well as the formation constants of weak Mg(j)H( i )(GSH)((i+2j-3)) and Ca(j)H(i)(GSH)((i+2j-3)) complexes. Direct calorimetric titrations were also carried out in pure NaCl((aq)) and in NaCl((aq))/CaCl(2(aq)) mixtures at different ionic strengths (0.25 ≤ I (c )/mol L(-1) ≤ 5.0) in order to determine the enthalpy changes for the protonation and complex formation equilibria in these media at T = 298.15 K. Results obtained are useful for the definition of glutathione speciation in any aqueous media containing the main cations of natural waters and biological fluids, such as Na(+), K(+), Mg(2+), and Ca(2+). Finally, this kind of systematic studies, where a series of ionic media (e.g., all alkali metal chlorides) is taken into account in the determination of various thermodynamic parameters, is useful for the definition of some trends in the thermodynamic behavior of glutathione in aqueous solution. 相似文献
3.
Liu SQ Jin H Zacarias A Srivastava S Bhatnagar A 《The Journal of biological chemistry》2001,276(15):11812-11820
The beta-subunit of the voltage-sensitive K(+) (K(v)) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of K(v)beta2 shows NADP bound in its active site. Here we report that K(v)beta2 displays a high affinity for NADPH (K(d) = 0.1 micrometer) and NADP(+) (K(d) = 0.3 micrometer), as determined by fluorometric titrations of the recombinant protein. The K(v)beta2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, the K(d)(NADPH) of Q214R was 10-fold greater than the wild-type protein. The K(d)(NADPH) was unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease in K(d)(NADPH), shifted the pK(a) of K(d)(NADPH) from 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of K(v)beta2-nucleotide complexes may be formed in vivo. 相似文献
4.
The basal Mg2(+)-dependent ATPase activity is not part of the (H(+)+K+)-transporting ATPase reaction cycle.
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H T Van der Hijden S Kramer-Schmitt E Grell J J de Pont 《The Biochemical journal》1990,267(3):565-572
Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the 'stretched-out' version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase. 相似文献
5.
In Bacillus subtilis, P protein is the noncatalytic component of ribonuclease P (RNase P) that is critical for achieving maximal nuclease activity under physiological conditions. P protein is predominantly unfolded (D) at neutral pH and low ionic strength; however, it folds upon the addition of sulfate anions (ligands) as well as the osmolyte trimethylamine N-oxide (TMAO) [Henkels, C. H., Kurz, J. C., Fierke, C. A., and Oas, T. G. (2001) Biochemistry 40, 2777-2789]. Since the molecular mechanisms that drive protein folding for these two solutes are different, CD thermal denaturation studies were employed to dissect the thermodynamics of protein unfolding from the two folded states. A global fit of the free-energy of TMAO-folded P protein versus [TMAO] and temperature yields T(S), DeltaH(S), and DeltaC(p) of unfolding for the poorly populated, unliganded, folded state (N) in the absence of TMAO. These thermodynamic parameters were used in the fit of the data from the coupled unfolding/ligand dissociation reaction to obtain the sulfate dissociation constant (K(d)) and the DeltaH and DeltaC(p) of dissociation. These fits yielded a DeltaC(p) of protein unfolding of 826 +/- 23 cal mol(-)(1) K(-)(1) and a DeltaC(p) of 1554 +/- 29 cal mol(-)(1) K(-)(1) for the coupled unfolding and dissociation reaction (NL(2) --> D + 2L). The apparent stoichiometry of sulfate binding is two, so the DeltaC(p) increment of ligand dissociation is 363 +/- 9 cal mol(-)(1) K(-)(1) per site. Because N and NL(2) appear to be structurally similar and therefore similarly solvated using standard biophysical analyses, we attribute a substantial portion of this DeltaC(p) increment to an increase in conformational heterogeneity coincident with the NL(2) --> N + 2L transition. 相似文献
6.
We report time-resolved optical measurements of the primary electron transfer reactions in Rhodobacter capsulatus reaction centers (RCs) having four mutations: Phe(L181) --> Tyr, Tyr(M208) --> Phe, Leu(M212) --> His, and Trp(M250) --> Val (denoted YFHV). Following direct excitation of the bacteriochlorophyll dimer (P) to its lowest excited singlet state P, electron transfer to the B-side bacteriopheophytin (H(B)) gives P(+)H(B)(-) in approximately 30% yield. When the secondary quinone (Q(B)) site is fully occupied, P(+)H(B)(-) decays with a time constant estimated to be in the range of 1.5-3 ns. In the presence of excess terbutryn, a competitive inhibitor of Q(B) binding, the observed lifetime of P(+)H(B)(-) is noticeably longer and is estimated to be in the range of 4-8 ns. On the basis of these values, the rate constant for P(+)H(B)(-) --> P(+)Q(B)(-) electron transfer is calculated to be between approximately (2 ns)(-)(1) and approximately (12 ns)(-)(1), making it at least an order of magnitude smaller than the rate constant of approximately (200 ps)(-)(1) for electron transfer between the corresponding A-side cofactors (P(+)H(A)(-) --> P(+)Q(A)(-)). Structural and energetic factors associated with electron transfer to Q(B) compared to Q(A) are discussed. Comparison of the P(+)H(B)(-) lifetimes in the presence and absence of terbutryn indicates that the ultimate (i.e., quantum) yield of P(+)Q(B)(-) formation relative to P is 10-25% in the YFHV RC. 相似文献
7.
An animal-vegetal net ionic current identified previously using voltage probe techniques in maturing Xenopus laevis oocytes has now been investigated using noninvasive ion-selective microelectrodes. Three-dimensional fluxes of hydrogen (H(+)), potassium (K(+)), and bicarbonate (HCO(3)(-)) were characterized with respect to the developmental stage and hemisphere of the oocyte and presence of surrounding follicular tissue. Variable effluxes of H(+) and HCO(3)(-) were recorded from both the animal and vegetal hemispheres. Variable influxes and effluxes of K(+) were also observed. The equatorial region, silent by voltage probe, exhibited fluxes of H(+) and K(+). Simultaneous measurement of pairs of ions allowed correlation analysis of two ion types. Notably for H(+) and K(+) data, positive and negative correlation at animal and vegetal poles respectively offer an explanation of the unpredictable results obtained when individual ions were observed independently. 相似文献
8.
Trobonjaca Z Leithäuser F Möller P Bluethmann H Koezuka Y MacDonald HR Reimann J 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(6):3804-3812
CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential. 相似文献
9.
A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate. 相似文献
10.
Lew RR 《Journal of experimental botany》2007,58(12):3475-3481
Voltage dependence of ionic currents and ion fluxes in a walled, turgor-regulating cell were measured in Neurospora crassa. The hyphal morphology of the model organism Neurospora simplifies cable analysis of ionic currents to determine current density for quantitative comparisons with ion fluxes. The ion fluxes were measured directly and non-invasively with self-referencing ion-selective microelectrodes. Four ions (H(+), Ca(2+), K(+), and Cl(-)) were examined. H(+) net uptake and Ca(2+) net release were small (10.2 nmol m(-2) s(-1) and 1.1 nmol m(-2) s(-1), respectively) and voltage independent. K(+) and Cl(-) fluxes were larger and voltage dependent. Maximal K(+) net release ( approximately 1440 nmol m(-2) s(-1)) was observed at positive voltages (+15 mV), while maximal Cl(-) net release ( approximately 905 nmol m(-2) s(-1)) was observed at negative voltage (-210 mV). A possible function of the net outward K(+) and Cl(-) fluxes is regulation of the plasma membrane potential. Total ion fluxes were 37-58% of the total ionic current density (about +/-244 mA m(-2), equivalent to +/-2500 nmol m(-2) s(-1), at 0 mV and -200 mV) so other ions must contribute significantly to the ionic currents. 相似文献
11.
The effect of the pH of an electrolyte solution on the electric surface charge of the liposome membrane was studied. The membrane of vesicles contained egg phosphatidylcholine (PC) with different proportions of stearylamine (ST). The surface charge density of the membrane was determined as a function of pH from electrophoretic mobility measurements. A six equilibria model describing the solution ions adsorption on the PC-ST liposome membrane surface was presented in this paper. The knowledge of the association constants of the -PO(-) and -N(+)CH(3)(3) groups of PC with H(+), OH(-), Na(+), Cl(-) ions: K(A(1)H), K(B(1)OH), K(A(1)Na), K(B(1)Cl), that had been presented earlier, allowed to determine the association constants of the -N(+)H(3) group of ST with OH(-) and Cl(-) ions: K(B(2)OH), K(B(2)Cl). The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the PC-ST liposomal membrane with the experimental data. 相似文献
12.
T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH(2)p) (100), clodronate (pCCl(2)p) (52), and etidronate (pC(OH)(CH(3))p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH(2)-CH(2)-NH(2))p), and alendronate (pC(OH)(CH(2)-CH(2)-CH(2)-NH(2))p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K(m) values and the rate of synthesis (k(cat)) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26+/-0.05 mM (0.28+/-0.05 s(-1)); clodronate, 0.54+/-0.14 mM (0.29+/-0.05 s(-1)); and etidronate, 4.3+/-0.5 mM (0.028+/-0.013 s(-1)). In the presence of GTP, and ATP or AppCCl(2)p the relative rate of synthesis of adenosine 5',5'-P(1),P(4)-tetraphosphoguanosine (Ap(4)G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH(2)p) was a very poor substrate for the synthesis of Ap(4)G. To our knowledge this report describes, for the first time, the synthesis of ATP beta,gamma-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases. 相似文献
13.
New, partially hydrolyzable synthetic analogues of lecithin, phosphatidyl ethanolamine, and phosphatidic acid 总被引:1,自引:0,他引:1
A F Rosenthal 《Journal of lipid research》1966,7(6):779-785
The synthesis of two new synthetic analogues of lecithin, two of phosphatidyl ethanolamine ("cephalin"), and one new phosphatidic acid analogue is described. They comprise one of each of the following types: the "isosteric" diether lecithin and cephalin analogues ROCH(2)CH(OR)- CH(2)CH(2)P(O) (O(-))OCH(2)CH(2)N(+)R'(3) (R = C(18)H(37); R' = H or CH(3)); and the "hydrocarbon" analogues of phosphatidic acid, lecithin, and cephalin, C(17)H(35)CH(2)CH(C(18)H(37))CH(2)P(O)(R) = (R'); [R = R' = OH; R = O(-), R' = OCH(2)CH(2)N(+)(CH(3))(3); and R = O(-), R' = OCH(2)CH(2)N(+)H(3)]. Infrared spectra and other properties of these compounds are described. 相似文献
14.
Photosynthetic reaction centers produce and export oxidizing and reducing equivalents in expense of absorbed light energy. The formation of fully reduced quinone (quinol) requires a strict (1:1) stoichiometric ratio between the electrons and H(+) ions entering the protein. The steady-state rates of both transports were measured separately under continuous illumination in the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides. The uptake of the first proton was retarded by different methods and made the rate-limiting reaction in the photocycle. As expected, the rate constant of the observed proton binding remained constant (7 s(-)(1)), but that of the cytochrome photooxidation did show a remarkably large increase from 14 to 136 s(-)(1) upon increase of the exciting light intensity up to 5 W/cm(2) (808 nm) at pH 8.4 in the presence of NiCl(2). This corresponds to about 20:1 (e(-):H(+)) stoichiometric ratio. The observed enhancement is linearly proportional to the light intensity and the rate constant of the proton uptake by the acceptor complex and shows saturation character with quinone availability. For interpretation of the acceleration of cytochrome turnover, an extended model of the photocycle is proposed. A fraction of photochemically trapped RC can undergo fast (>10(3) s(-)(1)) conformational change where the semiquinone loses its high binding affinity (the dissociation constant increases by more than 5 orders of magnitude) and dissociates from the Q(B) binding site of the protein with a high rate of 4000 s(-)(1). Concomitantly, superoxide is being produced. No H(+) ion is taken up, and no quinol is created by the photocycle which is operating in about 25% of the reaction centers at the highest light intensity (5500 s(-)(1)) and slowest proton uptake (3.5 s(-)(1)) used in our experiments. The possible physical background of the light-induced conformational change and the relationship between the energies of dissociation and redox changes of the quinone in the Q(B) binding sites are discussed. 相似文献
15.
The effects of olive mill waste compost and poultry manure on the availability and plant uptake of nutrients in a highly saline soil 总被引:1,自引:0,他引:1
The effects of a compost (produced from by-products of the olive oil industry) and a poultry manure on mineral ion solubility and exchangeability in a highly saline agricultural soil (electrical conductivity for a 1:5 soil:water extract=1.85 dS m(-1)) from Murcia (SE Spain) were studied. The organic amendments did not change significantly the soil electrical conductivity or the soluble Na(+), Ca(2+) or Mg(2+). Only soluble K(+) increased, due to the K(+) supplied by the amendments. The cation exchange capacity increased in treated soils, the exchange complex being mainly saturated with Ca(2+), Mg(2+) and K(+). However, Na(+) was not retained in the exchange sites, and the sodium absorption ratio remained low. The compost and manure increased markedly the shoot growth of the salt-tolerant Beta maritima L. (sea beet) and Beta vulgaris L. (sugar beet). For B. maritima, this seemed to be related to decreases in the shoot concentrations of Na(+) and Cl(-) and increases in K(+) and H(2)PO(4)(-). In the case of B. vulgaris, increases in shoot H(2)PO(4)(-) and B and, for manure-treated soil, a decrease in shoot Na(+) may have been involved. Cultivation of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) in the soil used previously for B. vulgaris indicated that the effects of the manure on tissue cation concentrations were longer-lasting than those of the compost. 相似文献
16.
Cumulant analysis of charge recombination kinetics in bacterial reaction centers reconstituted into lipid vesicles
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The kinetics of charge recombination between the primary photoxidized donor (P(+)) and the secondary reduced quinone acceptor (Q(B)(-)) have been studied in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides incorporated into lecithin vesicles containing large ubiquinone pools over the temperature range 275 K = T = 307 K. To account for the non-exponential kinetics of P(+) re-reduction observed following a flash, a new approach has been developed, based on the following assumptions: 1) the exchange of quinone between different vesicles is negligible; 2) the exchange of quinone between the Q(B) site of the RC and the quinone pool within each single vesicle is faster than the return of the electron from the primary reduced acceptor Q(A)(-) to P(+); 3) the size polydispersity of proteoliposomes and the distribution of quinone molecules among them result in a quinone concentration distribution function, P(Q). The first and second moments of P(Q) have been evaluated from the size distribution of proteoliposomes probed by quasi-elastic light scattering (mean radius, = (50 +/- 15) nm). Following these premises, we describe the kinetics of P(+)Q(B)(-) recombination with a truncated cumulant expansion and relate it to P(Q) and to the free energy changes for Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer (DeltaG(AB)(o)) and for quinone binding (DeltaG(bind)(o)) at Q(B). The model accounts well for the temperature and quinone dependence of the charge recombination kinetics, yielding DeltaG(AB)(o) = -7.67 +/- 0.05 kJ mol(-1) and DeltaG(bind)(o) = -14.6 +/- 0.6 kJ mol(-1) at 298 K. 相似文献
17.
The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-)(1), K(d) = 37 microM) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings. 相似文献
18.
Irving M Zoete V Hebeisen M Schmid D Baumgartner P Guillaume P Romero P Speiser D Luescher I Rufer N Michielin O 《The Journal of biological chemistry》2012,287(27):23068-23078
Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ~1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ~1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies. 相似文献
19.
The ONIOM(B3LYP/6-31G(d):AM1) optimized structures of complexes of diurea calix[4]arene receptor (L) with alkali metals Li(+), Na(+) and K(+) and their complexes with halide ions F(-), Cl(-), Br(-), oxygen-containing anions HCO(3)(-), HSO(4)(-) and CH(3)COO(-) ions were obtained. Binding energies and thermodynamic properties of complex receptors LiL(+), NaL(+) and KL(+) with these anions were determined. The binding stabilities according to binding energies of LiL(+), NaL(+) and KL(+) associated with anions computed either at the ZPVE-corrected ONIOM(B3LYP/6-31G(d):AM1) or BSSE-corrected B3LYP/6-31 + G(d,p)//ONIOM(B3LYP/6-31G(d):AM1) are in the same order: F(-) > CH(3)COO(-) ≈ HCO(3)(-) > Br(-) ≈ HSO(4)(-) ≈ Cl(-). All the receptors LiL(+), NaL(+) and KL(+) were found to be selective toward fluoride ion. 相似文献
20.
Kuklin NA Rott L Feng N Conner ME Wagner N Müller W Greenberg HB 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(3):1894-1902
Rotavirus (RV) is the main cause of severe gastroenteritis in young children; protection has been correlated with intestinal Ab responses. Using a mouse model of RV infection and beta(7)-deficient (beta(7)(-/-)) mice, which do not express alpha(4)beta(7) integrin, we demonstrated the importance of alpha(4)beta(7) integrin in B cell-mediated anti-RV immunity. beta(7)(-/-) mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. alpha(4)beta(7)(-/-) immune B cells did not resolve RV infection when adoptively transferred into RV-infected Rag-2-deficient mice. Fewer RV-specific B cells were found in the intestine of Rag-2-deficient mice transferred with beta(7)(-/-) B cells compared with wild type. The absence of alpha(4)beta(7) expression and/or a lower frequency of IgA-producing cells among transferred beta(7)(-/-) B cells could have accounted for the inability of these cells to resolve RV infection following passive transfer. To distinguish between these possibilities, we studied the importance of IgA production in RV infection using IgA-deficient (IgA(-/-)) mice. IgA(-/-) mice depleted of CD8(+) T cells were able to clear primary RV infection. Similarly, adoptive transfer of immune IgA(-/-) B cells into chronically infected Rag-2-deficient mice resolved RV infection. We further demonstrated in both wild-type and IgA(-/-) mice that, following oral RV infection, protective B cells reside in the alpha(4)beta(7)(high) population. Our findings suggest that alpha(4)beta(7) integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA. 相似文献