共查询到20条相似文献,搜索用时 31 毫秒
1.
pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method. 总被引:4,自引:0,他引:4
On the basis of previous stopped-flow pH-jump experiments, we have proposed that the acid- and alkaline-induced folding/unfolding transition of staphylococcal nuclease, in the time range 2 ms to 300 s, follows the pathway N0 in equilibrium with D1 in equilibrium with D2 in equilibrium with D3, in which D1, D2, and D3 are three substates of the unfolded state and N0 is the native state. The stopped-flow "double-jump" technique has been employed to test this mechanism and to determine the rate constants which would not be accessible by the direct pH jump because of the lack of fluorescence signal, i.e., the rates for the conversion of D1 to D2 and of D2 to D3. In the forward jump, a protein solution kept at pH 7.0 was mixed with an acidic or alkaline solution to the final pH of 3.0 or 12.2, respectively. The mixed solution was kept for varying periods of time, called the delay time, tD. A second mixing (the back jump) was launched to bring the protein solution back to pH 7.0. The time course of the Trp-140 fluorescence signals recovered in the back jump was analyzed as a function of tD. Kinetics of the unfolding were found to be triphasic by the double-jump method, contrary to the monophasic kinetics observed by the direct pH jump. Complex kinetics of unfolding are expected with the proposed kinetic scheme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
The kinetics of the electrostatically induced phase transition of dimyristoyl phosphatidic acid bilayers was followed using the stopped-flow technique. The phase transition was triggered by a fast change in the pH or the magnesium ion concentration and followed by recording the time dependence of the absorbance. When the phase transition was induced by a pH jump the time course of the absorbance could be described by two exponentials, their time constants displaying the for cooperative processes characteristic maximum at the transition midpoint. The time constants are in the 10 and 100 ms range for the H+ triggered transition from the fluid to the ordered state. A third slower process shows no appreciable temperature dependence and is probably caused by vesicle aggregation. For the OH--induced transition fron the ordered to the fluid state the time constants are in the 100 and 1000 ms range. The fluid-ordered transition could also be triggered by addition of magnesium ions. Of the several observed processes only the fastest in the 10–100 ms time range could definitely be assigned to the fluid-ordered transition while the others are due to aggregation phenomena. The experimental data were compared with results obtained from pressure jump experiments and could be interpreted on the basis of theories for non-equilibrium relaxation. 相似文献
3.
A kinetic mechanism is presented for Escherichia coli dihydrofolate reductase which describes the full time course of the enzymatic reaction over a wide range of substrate and enzyme concentrations at pH 7.2 and 20 degrees C. Specific rate constants were estimated by computer simulation of the full time course of single turnover, burst, and steady-state experiments using both nondeuterated and deuterated NADPH. The mechanism involves the random addition of substrates, but the substrates and enzyme are not at equilibrium prior to the chemical transformation step. The rate-limiting step follows the chemical transformation, and the maximum velocity of the reaction is limited by the release of the product tetrahydrofolate. The full time course of the reaction is markedly affected by the formation of the enzyme-NADPH-tetrahydrofolate abortive complex, but not by the enzyme-NADP-dihydrofolate abortive complex. 相似文献
4.
The thickness changes of black lipid membranes of different composition after a voltage jump were investigated. In a second series of electrical relaxation experiments the kinetics of channel formation by gramicidin A were measured. The time course of the membrane current was compared with the time course of the thickness change of the membranes. We found that the time course of the current as a consequence of channel formation by gramicidin A did not correlate with the thickness change of the lipid membranes. A possible direct influence of the electric field is discussed. 相似文献
5.
Aggregation of the Alzheimer's disease-related Aβ1-28 peptide was induced by a rapid, sub-millisecond pH jump and monitored by time-resolved infrared spectroscopy on the millisecond to second time-scale. The release of protons was induced by the photolysis of a caged compound, 1-(2-nitrophenyl)ethyl sulfate (NPE-sulfate). The pH jump generated in our experimental setup is used to model the Aβ peptide structural conversions that may occur in the acidic endosomal/lysosomal cell compartment system. The aggregation of the Aβ1-28 peptide induced by the pH jump from 8.5 to < 6 yields an antiparallel β-sheet structure. The kinetics of the structural transition is biphasic, showing an initial rapid phase with a transition from random coil to an oligomeric β-sheet form with a time constant of 3.6 s. This phase is followed by a second slower transition, which yields larger aggregates during 48.0 s. 相似文献
6.
K. Lívanský 《Folia microbiologica》1981,26(5):422-425
In a culture ofChlorella kessleri illuminated with a sodium vapour lamp at 33 °C and pH 6.5 the maximum rate of biomass production was 4.5 g L-l d-1. Of the total volume of 3.6 L of the suspension 2.5 L were placed between two glass concentric cylinders surrounding the tube. Examples of the course of speoific growth rate μ in the transient state following jump changes of the dilution rate, pH and temperature are presented. After a jump change of pH or temperature of the suspension maximal and minimal values, respectively, of μ are observed. 相似文献
7.
Mitochondria, uncoupled by aging or by freeze-thaw treatment, are able to synthesize ATP from ADP and Pi after a fast increase (but not decrease) in the external pH. The maximal ATP yield (approx. 2.5 ATP molecules / electron-transport chain per pH jump) can be obtained under the following conditions: (1) the pH change during the jump must exceed 0.7 pH units; (2) in the course of this change, the pH of the mitochondrial suspension must cross the pH 8.1–8.3 value. This pH-jump-induced ATP synthesis is completely inhibited by oligomycin. 相似文献
8.
L A Blumenfeld 《Journal of biomolecular structure & dynamics》1988,6(1):23-33
Membrane-bound and isolated H+ ATPases of various origin are able to synthesize ATP from ADP and Pi after a jump-like pH increase. In the course of this increase the pH of solution (or suspension) must cross a value corresponding to pK of certain acid groups in the catalytic component of ATPase. In the case of isolated soluble enzymes it is possible to obtain up to 10 ATP molecules per one pH jump per one enzyme molecule. A physical mechanism of this phenomenon as well as of oxidative and photosynthetic phosphorylation is suggested. 相似文献
9.
Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin.
A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins. 相似文献
10.
Determination of the rate of rapid pH equilibration across isolated sarcoplasmic reticulum membranes
The rate of the transmembrane pH equilibration in the isolated vesicles of sarcoplasmic reticulum of skeletal muscle after extravesicular pH jump was determined for the first time. A highly water-soluble pH sensitive fluorescent dye, 8-hydroxy-1,3,6-pyrenetrisulphonic acid (pyranine), was used as intravesicular pH indicator in the stopped-flow fluorophotometry. The pH of the medium was controlled with 20 mM HEPES-K or PIPES-K. The fluorescence intensity changed monophasically as much as expected from its pH dependency for imposed delta pH. The half time for initial pH of 7.53 or 6.83 was about 6 msec. The H+/OH- permeability was 11 cm/sec. The results suggested that each vesicle contained large numbers of H+/OH- channels. 相似文献
11.
Properties of the dolichol phosphate: GDPmannose mannosyltransferase of liver microsomes 总被引:1,自引:0,他引:1
The reaction of GDP[14C]-mannose with dolichol phosphate (Dol-P) in hepatic microsomes is characterized by an initial brief period of relatively rapid Dol-P-[14C]-mannose synthesis. The time course of this 1--3 min period of rapid synthesis follows approximate first order kinetics. However, the rate of reaction does not decrease to zero as predicted by the kinetics of the initial period of synthesis, but continues instead at a slow, steadily decreasing, rate. Examination of the time course of Dol-P-mannose synthesis for different concentrations of GDP[14C]-mannose revealed that the extrapolated final level of Dol-P-mannose synthesized is increased when the concentration of GDPmannose is raised. These data, plus those derived from studies of the reverse reaction, suggest that the non-linear time course for the synthesis of Dol-P-mannose is due in part to the reaction approaching equilibrium between the forward and reverse reactions. The effects of Mn++ on the time course of the forward and reverse reaction are complex and suggest that the Mn++ complexes of both GDPmannose and GDP are poorer substrates for the enzyme than the free nucleotides. Perturbations of the lipid environment of the microsomal membrane by treatment with phospholipase A, detergent, sonication, or alkaline pH lead to a decrease in the final level of Dol-P-mannose synthesized, but do not affect the time required for half maximal labeling. When the reverse reaction was investigated in phospholipase A-treated microsomes, the final extent of the reaction was also reduced. These data suggest that perturbation of the membrane lipid environment decreases in some undefined way the availability of Dol-P and Dol-P-mannose to enzyme. 相似文献
12.
Participation of H+ in the Ca2(+)-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase 总被引:1,自引:0,他引:1
The binding of Ca2+ to 4-nitro-2,1,3-benzoxadiazole (NBD)-labeled sarcoplasmic reticulum Ca2(+)-ATPase was accelerated markedly when the pH was changed at 11 degrees C from 6.5 to 8.0 at the time of Ca2+ addition. We examined the effect of pH on the enzyme conformational transition by measuring the kinetics of NBD fluorescence rises induced by a pH jump under various ligand conditions. The fast fluorescence rise following a pH jump from 6.0 or 6.5 to various test pHs in the presence and absence of Ca2+ proceeded monoexponentially. The amplitude of this fluorescence rise in the presence of Ca2+ was independent of the test pH, whereas the observed rate constant (kobs) increased markedly as the test pH increased. In contrast, the amplitude of the fast fluorescence rise in the absence of Ca2+ increased with increasing test pH, whereas kobs decreased. MgATP or Mg2+ influenced the pH dependences of these parameters in a complex way except for the amplitudes measured in the presence of Ca2+. These data could be simulated by using a reaction model in which Ca2+ binding is preceded by a rate-limiting enzyme conformational transition from a low to a high NBD fluorescence state and 1 mol each of H+ is liberated before and after this conformational transition. MgATP or Mg2+ appeared to promote this conformational transition by enhancing deprotonation of the enzyme. These results suggest that deprotonation may be the primary event in the activation of the unphosphorylated enzyme by Ca2+. 相似文献
13.
The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated to be 760 and 4. 7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the folding rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same as the rate for the domain in the intact protein. 相似文献
14.
《Inorganica chimica acta》1986,125(2):97-106
We report studies on the interaction of iron(III) and ascorbic acid as a function of pH in pure water, pure methanol and mixtures of these solvents.Mössbauer data indicates the iron(III) is reduced in water at low pH to iron(II). Rapid mixing studies and pH jump investigations using stopped flow spectrophotometry have been used to follow the reactions and show evidence for blue intermediates in the reduction pathway of iron at low pH values. A scheme is proposed to account for the complex reaction between iron and ascorbate in aqueous solvent. Binding constants between iron(III) and ascorbate are given. 相似文献
15.
Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods. 相似文献
16.
Stefania Abbruzzetti Silvia Sottini Cristiano Viappiani John E T Corrie 《Photochemical & photobiological sciences》2006,5(6):621-628
Using 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) as a photoactivatable caged proton, we could induce complete acid unfolding of myoglobin with a single nanosecond laser pulse. This was possible because of the high ( approximately mM) concentration of protons released by the photolabile compound. The ability of the compound to produce a large pH jump arises because the other photoproducts (2-nitrosoacetophenone and sulfate ion) do not buffer the released protons. The complete time course of the unfolding kinetics, spanning a range from milliseconds to several seconds, could be accurately reproduced by monitoring absorbance changes in the visible spectrum at 633 nm. 相似文献
17.
The beta-lactamase from Shigella flexneri UCSF-129 was irreversible inactivated by 6-beta-iodopenicillanic acid. Only one serine residue was modified, according to the spectra change and the amino acid analyses. A pH variation of 0.3 units was found when the chemically modified enzyme was submitted to isoelectric focusing. The inactivation constant of the fast time course reaction was 0.1 seg-1. Protection of 96% was obtained, using cephradine 2,830 times more concentrated than 6-beta-iodopenicillanic acid. It is suggested that this enzyme belongs to class A, according to Ambler (1980). 相似文献
18.
Stopped-flow studies of spectral changes in bilirubin-human serum albumin following an alkaline pH jump and following binding of bilirubin 总被引:1,自引:0,他引:1
B Honoré 《The Journal of biological chemistry》1987,262(31):14939-14944
A stopped-flow technique was used to study the spectral changes occurring in bilirubin-albumin following a pH jump as well as following binding of bilirubin at 25 degrees C. The changes were studied in two wavelength ranges, 280-310 nm (tyrosine residues) and 400-510 nm (bound bilirubin). The changes were analyzed according to a scheme of consecutive unimolecular reactions. Spectral monitoring of a pH jump from 11.3 to 11.8 reveals that the bilirubin-albumin complex changes its structure in several steps. The UV absorption spectra show that 3.8 tyrosine residues ionize in the first step, 2.5 in the second, none in the third, and 0.8 in the fourth and following steps. The visible absorption spectrum of bound bilirubin changes in the second, third, and fourth steps. The bilirubin spectra of the different bilirubin-albumin complexes occurring in the transition show a common isosbestic point at 445 nm, indicating a change of the dihedral angle between the two bilirubin chromophores in a three-step reaction. It is suggested that 1 tyrosine residue is located close to the bilirubin site and is externalized in the second step. Bilirubin binding to albumin was monitored at two pH values, 11.3 and 11.8. At pH 11.3 the complex changes its structure in a three-step relaxation sequence. A change of the dihedral angle between the bilirubin chromophores can explain the spectral changes observed in the second and third relaxations. Protonation of 0.7 tyrosine residues occurs in the third relaxation, suggesting internalization of a tyrosine residue as a late consequence of bilirubin binding. At pH 11.8 a two-step relaxation sequence follows bilirubin binding. No tyrosine protonation occurs. Bilirubin is probably bound more superficially at pH 11.8 than at pH 11.3. 相似文献
19.
A sudden pH decrease (pH jump) of the medium enhanced pyruvate uptake in the dark in mesophyll chloroplasts (MCp) of Zea mays and Sorghum bicolor, NADP-malic enzyme type C4 plants, while it was reported that a Na+ jump enhanced pyruvate uptake in MCp of P. miliaceum, a NAD-malic enzyme type [(1987) FEBS Lett. 219, 347]. The enhancement effect of the pH jump decayed completely in 5 min and the decay was accelerated by proton gradient-collapsing reagents. The results suggest that active pyruvate uptake into MCp of NADP-malic enzyme type C4 species is primarily driven by the proton gradient across the envelope. 相似文献
20.
In Monte Carlo simulations of water radiolysis, the diffusion of reactants can be approximated by “jumping” all species randomly,
to represent the passage of a short period of time, and then checking their separations. If, at the end of a jump, two reactant
species are within a distance equal to the reaction radius for the pair, they are allowed to react in the model. In principle,
the possibility exists that two reactants could “jump through” one another and end up with a separation larger than the reaction
radius with no reaction being scored. Ignoring this possibility would thus reduce the rate of reaction below that intended
by such a model. By making the jump times and jump distances shorter, any error introduced by `jump through' is made smaller.
This paper reports numerical results of a systematic study of `jump through' in Monte Carlo simulations of water radiolysis.
With a nominal jump time of 3 ps, it is found that more than 40% of the reactions of the hydrated electron with itself and
of the H atom with itself occur when reactions during `jump through' are allowed. For all other reactions, for which the effect
is smaller, the contributions of `jump through' lie in the range l%–16% of the total. Corrections to computed rate constants
for two reactions are evaluated for jump times between 0.1 and 30 ps. It is concluded that jump-through corrections are desirable
in such models for jump times that exceed about 1 ps or even less. In a separate study, we find that giving all species of
a given type the same size jump in a random direction yields results that are indistinguishable from those when the jump sizes
are selected from a Gaussian distribution. In this comparison, the constant jump size is taken to be the root-mean-square
jump size from the Gaussian distribution.
Received: 8 September 1997 / Accepted in revised form: 27 October 1997 相似文献