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1.
31P NMR studies of intact cells and perchloric acid extracts are used to investigate the effect of ethanol on the bioenergetics and glycolysis of Clostridium thermocellum, an anaerobic bacterium potentially useful for the single step conversion of biomass to ethanol. Whole cells suspended in phosphate buffer and given a carbon source (cellobiose) at 60 degrees C rapidly establish a pH gradient across the membrane that can be monitored by the chemical shifts of inorganic phosphate in the exterior buffer and in the cytoplasm. Peak intensities can be related to phosphate active transport rates. Wild type bacteria and cells grown in inhibiting concentrations of ethanol establish similar pH gradients, but with slower kinetics and slower phosphate transport rates for the cells adapted to growth in ethanol. Direct addition of ethanol does not affect the rate of pH gradient formation or phosphate transport. Thus, while ethanol does not directly affect processes for energy conservation carried out by the membrane, adaptation to ethanol does alter membrane functions such as phosphate transport. 31P NMR spectra of perchloric acid extracts show that when wild type cells are adapted to grow in inhibiting concentrations of ethanol and then energized with cellobiose, sugar phosphate content is increased and the steady state distribution of glycolytic intermediates is altered. Nucleotide triphosphate/nucleotide diphosphate ratios are unaltered in these cells. These results strongly indicate that in C. thermocellum growth inhibition by ethanol is related to a blockage in glycolysis.  相似文献   

2.
Phosphate transport in Micrococcus lysodeikticus.   总被引:3,自引:0,他引:3  
Phosphate accumulates in Micrococcus lysodeikticus cells against a concentration gradient, by an energy-dependent process. The phosphate transport is derepressed during phosphate deprivation. The depression process is inhibited by chloramphenicol. The apparent Km of phosphate transport is 4.3 micronM. The activation energy of the transport is 21 kcal per mol in the temperature range of 0-29degrees C, and 4.9 kcal per mol between 29 and 40degrees C. The rate of the transport increases in presence of K+ and Mg2+. Arsenate is a competitive inhibitor of phosphate transport, having an apparent Ki of 6.0 micronM. Sulfhydryl reagents, respiratory inhibitors and uncouplers of oxidative phosphorylation inhibit phosphate transport.  相似文献   

3.
Growing cultures of Streptococcus faecalis at temperatures above 30 C have activation energies for both rates of growth and glycolysis of 10.3 kcal mole(-1), and a constant growth yield; when growth takes place below this temperature, the growth yield decreases and the activation energy for growth increases to 21.1 kcal mole(-1), but the activation energy for glycolysis is unchanged. The adenosine triphosphate pool in the organisms behaves differently above and below 30 C, suggesting that the energetic coupling between anabolism and catabolism is less effective below 30 C. Washed suspensions of S. faecalis have repressed glycolytic activity and an activation energy for glycolysis of 15.6 kcal mole(-1) over the whole temperature range studied. Growing cultures of Zymomonas mobilis below 33 C have a constant growth yield of 8.3 g (dry weight) of organisms per mole of glucose degraded, and activation energies for both glycolysis and growth of 11.1 kcal mole(-1); above this temperature, the growth yield falls, the activation energy for growth changes to -6.9 kcal mole(-1), but the activation energy for glycolysis is unchanged, so that the coupling between anabolism and catabolism is less effective above 33 C. The findings support the view that energy turnover in these bacteria is not well regulated.  相似文献   

4.
Energetics of the calcium-transporting ATPase   总被引:11,自引:0,他引:11  
A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.  相似文献   

5.
Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.  相似文献   

6.
The cellular autolytic reaction system in Streptococcus faecalis ATCC 9790 was analyzed for relative increases in reaction rates with increasing temperature by determination of Arrhenius activation energies (E). The systems examined were: (i) an isolated wall-enzyme complex in 0.01 M sodium phosphate, pH 6.9; (ii) exponential-phase cells suspended in 0.01 or o.3 M sodium phosphate pH 6.8, or in 0.04 M ammonium acetate, pH 6.8, (iii) growing cultures deprived of glucose or lysine; and (iv) cultures treated in growth media with the nonionic detergent, Triton X-100. For detergent-treated cells, E values were between 23.9 and 27.4 kcal/mol (ca. 100.1 to 174.7 kJ/mol) at concentrations of Triton X-100 between about 0.03 and 0.072 mg/ml. E values dropped sharply to 11.5 to 13.0 kcal/m-l (ca. 48.2 to 54.4 kJ/mol) at Triton X-100 concentrations of 0.12 mg/ml or higher. For the remaining systems, E values ranged from 16 to 20 kcal/mol (ca. 67.0 to 83.7 kJ/mol) (wall lysis, cellular autolysis in 0.01 M sodium phosphate or in 0.04 M ammonium acetate, and autolysis of glucose-starved cells) to 31 to 38 kcal/mol (ca 129.8 to 159.1 kJ/mol) (cellular autolysis in 0.3 M sodium phosphate or autolysis of lysine-starved cells). High concentrations of Triton X-100 appear to lower the E values below the 16 to 20 kcal/mol observed for the autolysis of isolated walls. This effect may be related to disruption by the detergent of a hydrophobic complex regulating cellular autolysis in vivo.  相似文献   

7.
T4 DNA ligase is an Mg2+-dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a 'processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a 'nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (<10 microM) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (approximately 0.2 min(-1)) and a subsequent slow ligation (approximately 2x10(-3) min(-1)). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal.mol(-1)), transadenylation of the nick (0.9 kcal.mol(-1)), and nick-sealing (16.3-18.8 kcal.mol(-1)) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis.  相似文献   

8.
Thymidylate kinase derived from the blast cells of human chronic myelocytic leukemia was purified 2186-fold to near homogeneity by means of alcohol precipitation, alumina-Cgamma gel fractionation, calcium phosphate gel fraction, ultrafiltration, and affinity column chromatography. The molecular weight was estimated by glycerol gradient centrifugation to be 50,000. This enzyme had an optimal activity at pH 7.1 and required a divalent cation in order to catalyze the reaction. Mg2+ and Mn2+ were found to be the preferential divalent cations. The activation energy was estimated to be 19.1 kcal/mol at pH 7.2. Initial velocity study suggested that the reaction followed a sequential mechanism. Mg2+ ATP had a Km of 0.25 mM and dTMP had a Km of 40 micrometer. The enzyme was unstable even at 4 degrees. In the presence of ATP or dTMP the enzyme maintained its activity. Purine triphosphate nucleosides were found to be better phosphate donors than the pyrimidine triphosphate nucleosides. ATP and dATP had a lower Km and a higher Vmax than GTP and dGTP. dTMP was the only preferred phosphate receptor among all the monophosphate nucleotides tested dTTP and IdUTP competed with both substrates and inhibited the reaction with a Ki of 0.75 mM and 1.1 mM, respectively.  相似文献   

9.
The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.  相似文献   

10.
The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the energy derived from ATP hydrolysis. During catalysis, part of the energy is used to translocate Ca(2+) across the membrane, and part is dissipated as heat. At 35 degrees C the heat released during the hydrolysis of each ATP molecule varies depending on the formation of a Ca(2+) gradient across the membrane. With leaky vesicles (no gradient) the heat released varies between 9 and 12 kcal/mol of ATP cleaved, and with intact vesicles (gradient), the heat released increases to 20-24 kcal/mol of ATP. After Ca(2+) accumulation, 82% of the Ca(2+)-ATPase activity is not coupled to Ca(2+) transport, and the ratio between Ca(2+) transported and ATP cleaved is 0.3. The addition of 20% dimethyl sulfoxide (v/v) to the medium or decreasing the temperature from 35 to 20 degrees C abolishes the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient. This is accompanied by a simultaneous inhibition of the uncoupled ATPase activity and an increase of the Ca(2+)/ATP ratio from 0.3 to 1.3-1.4. It is concluded that the uncoupled Ca(2+)-ATPase is responsible for both the low Ca(2+)/ATP ratio measured during transport and the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient.  相似文献   

11.
Uptake of phosphate by Streptococcus lactis ML3 proceeds in the absence of a proton motive force, but requires the synthesis of ATP by either arginine or lactose metabolism. The appearance of free Pi internally in arginine-metabolizing cells corresponded quantitatively with the disappearance of extracellular phosphate. Phosphate transport was essentially unidirectional, and phosphate concentration gradients of up to 10(5) could be established. Substrate specificity studies of the transport system indicated no preference for either mono- or divalent phosphate anion. The activity of the phosphate transport system was affected by the intracellular Pi concentration by a feedback inhibition mechanism. Uncouplers and ionophores which dissipate the pH gradient across the cytoplasmic membrane inhibited phosphate transport at acidic but not at alkaline pH values, indicating that transport activity is regulated by the internal proton concentration. Phosphate uptake driven by arginine metabolism increased with the intracellular pH with a pKa of 7.3. Differences in transport activity with arginine and lactose as energy sources are discussed.  相似文献   

12.
Nickel transport in Methanobacterium bryantii.   总被引:10,自引:4,他引:6       下载免费PDF全文
Methanobacterium bryantii, grown autotrophically on H2-CO2, transported nickel against a concentration gradient by a high-affinity system (Km = 3.1 microM). The system had a pH optimum of 4.9 and a temperature optimum of 49 degrees C with an energy of activation of 7.8 kcal/mol (ca. 32.6 kJ/mol). A headspace of H2-CO2 (4:1, vol/vol) was required for maximum rate of transport. The system was highly specific for nickel and was unaffected by high levels of all monovalent and divalent ions tested (including Mg2+) with the sole exception of Co2+. Kinetic experiments indicated that accumulated nickel became increasingly incorporated into cofactor F430 and protein. Nickel transport was inhibited by nigericin, monensin, and gramicidin but not by carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, N,N'-dicyclohexylcarbodiimide, valinomycin plus potassium, or acetylene. The ineffectiveness of carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, and N,N'-dicyclohexylcarbodiimide may be related to difficulties in the penetration of these compounds through the outer cell barriers. Nickel uptake was greatly stimulated by an artificially imposed pH gradient (inside alkaline). The data suggest that nickel transport is not dependent on the membrane potential or on intracellular ATP, but is coupled to proton movement.  相似文献   

13.
Stability of acqueous solutions of 6-beta-[(hexahydro-IH-azepin-I-yl)methylenamino] penicillanic acid at various values of pH and temperature was studied. It was found that inactivation of the antibiotic in both the acid and the alkaline medium proceeded according to the equation of the 1st order. At pH 1.3 and a temperature of 35 degrees the half life of the antibiotic was 7 hours. The activation energy calculated according to the Arrenius equation was 13.5 kcal/mol at pH 1.3 and 22.2 kcal/mol at pH 10.5. The antibiotic was inactivated in glycol and phosphate buffers. Its qualitative analysis was performed according to an improved iodometric method.  相似文献   

14.
Temperature jump and stopped flow methods were used to study at pH 7.0 the temperature dependence of elementary steps of the reactions of lysozyme with the beta(1 yields 4)-linked trimer, tetramer, and hexamer of N-acetylglucosamine. The steady state rate of cleavage of the hexasaccharide was determined as a function of temperature (5 degrees-40 degrees) and pH(2 to 8) in H-2O solution and as a function of pD(2.5 to 9.5) at 40 degrees in D-2O solution. The apparent enthalpies of the two ionizations of apparent pK 3.8 and 6.7 observed in measurements of k are 0 to 2 kcal/mol. The energy of activation determined for the pH optimum is 21.5 kcal/mol. The solvent deuterium isotope effect measured for k at the pH (pD) optimum is 1.5 And reflects isotope effects on pre-equilibrium steps and on the rate-determining step. Transfer from H-2O to D-2O solution produces 0.2 to 0.4 kcal/mol more negative free energies of saccharide binding and no changes in the enthalpies of binding. Pre-steady state, steady state, and equilibrium measurements indicate a pathway for the reaction of lysozyme with hexasaccharide. The results define for this mechanism the complete free energy profile and an essentially complete enthalpy profile. Three of the five observable ES complexes are present at nearly equal concentrations. The free energies of the transition states are within a range of 3 kcal. The enthalpies of productive enzyme-substrate complexes are about 5 kcal/mol greater than the enthalpies of nonproductive complexes. Changes in tryptophan fluorescence were observed for each elementary step, and changes in pK of Glu-35 for the isomerizations of nonproductive and productive complexes. The signal changes during formation of nonproductive complexes are the same for the oligosaccharides (ClcNAc)3 to (GlcNAc)6. The changes for productive complexes are similar but not identical with saccharides (GlcNAc)4 to (GlcNAc)6. Correlations of the present data with previous crystallographic and solution measurements indicate the structures of productive and nonproductive ES complexes and suggest that full interaction of the substrate with the enzyme active site is established in the rate-determining step.  相似文献   

15.
We studied the effect of compounds that uncouple oxidative phosphorylation on membrane function in Streptoccocus faecalis, an organism which relies upon glycolysis for the generation of metabolic energy. At low concentrations (ranging from 10(-7) to 10(-4)m), tetrachlorosalicylanilide, tetramethyldipicrylamine, carbonylcyanide m-chlorophenylhydrazone, pentachlorophenol, and dicoumarol strongly inhibited energy-dependent transport of rubidium, phosphate, and certain amino acids. However, these compounds had little effect on the generation of adenosine triphosphate via glycolysis or on its utilization for the synthesis of macromolecules. They also did not seriously inhibit uptake of those monosaccharides and amino acids which do not require concurrent metabolism. It is proposed that the uncouplers interfere with the utilization of metabolic energy for membrane transport. The uncouplers accelerated the translocation of protons across the cytoplasmic membrane. It appears that a proton-impermeable membrane is required for transport, perhaps, because a proton gradient is involved in the coupling of metabolic energy to the translocation of substrates across the membrane.  相似文献   

16.
The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.  相似文献   

17.
The completely hydrolyzed titanocene dichloride, [Cp2Ti(H2O)2]2+ binding to guanine (G) and phosphate group sites of DNA were investigated by DFT method, with using deoxyguanosine monophosphate (dGMP) as incoming ligand. In the first substitutions, the calculations reveal that the diaquated titanocene binding to O6 shows the lowest activation free energy with 17.9 kcal/mol, closely followed by N7 is 20.5 kcal/mol and the O of phosphate group is 26.3 kcal/mol, respectively. It was also found that all the titanation processes are mildly endothermic. In addition, for the Ti-B(dGMP) in all separated products, the bond dissociation free energies (BDFE) of Ti-O(P, P = phosphate) is higher than those of Ti-N7/O6. In the second substitutions, the reactions leading to the didentate adducts are considered. For bidentate-bridging N7, O6 binding mode, the path of the metal Ti binding to O6 has the lower activation free energy (11.3 kcal/mol) than that of the metal Ti binding to N7 (15.3 kcal/mol). For the bidentate-bridging N7, O(P) binding mode, the path of the metal Ti binding to O(P) has the lower activation free energies (25.3 kcal/mol) than that of the metal Ti binding to N7 (26.2 kcal/mol).  相似文献   

18.
Sarcoplasmic reticulum (SR) fragments from the skeletal muscles of rabbit with marked atherosclerosis possessed decreased Ca2+-accumulating capacity. Lowering of transport efficiency, namely reduction of the Ca/ATP ratio from 1.9--normal value--to 0.9 during the experiment at 26 degrees C was accompanied by activation of Ca-ATPase and simultaneously of the rate of Ca2+ outflux from the SR. Arrhenius plots of Ca-ATPase temperature dependence characterized under normal conditions by a break at 20--21 degrees C was linearized under hypercholesterolemia. At the same time there was a rise (from 0.03 under normal conditions to 0.15 in atherosclerosis) of cholesterol/protein ratio in the SR membrane preparations. Activation energy for Ca-ATPase crude membranes under normal conditions was equal to 15.6 and 28.7 kcal/mol above and below the break point respectively; this value for Ca-ATPase of membranes with increased cholesterol level was 19 kcal/mol for all the temperatures investigated.  相似文献   

19.
Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.  相似文献   

20.
Assays of intracellular ATP, ADP, and inorganic phosphate allowed calculation of the phosphorylation potential (delta G'ATP/F) maintained during glycolysis by Streptococcus lactis. At the same time, the electrochemical H+ gradient (delta mu-H+/F) was evaluated by distribution methods, using radioactive tetraphenylphosphonium bromide as a probe for the membrane potential and salicylic acid as a probe for the pH gradient. Detailed comparisons were made at pH 5, when the reaction mediated by the proton-translocating ATPase (BF0F1) was likely to have been poised near equilibrium; for those conditions, the ratio delta G'ATP/delta mu-H+ was used to estimate stoichiometry for BF0F1 during ATP hydrolysis. At an external pH of 5, in the presence or absence of valinomycin, this ratio was close to 3, over a range of 370 to 510 mV (8.5 to 11.7 kcal/mol) for delta G'ATP/F and a range of 128 to 167 mV for delta mu-H+/F. Other work suggested that delta G'ATP/delta mu-H+ increased from its minimum value of 3 to 4.3 as the external pH changed from pH 5 to 7.  相似文献   

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