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1.
Charles F Fowler  Bessel Kok 《BBA》1976,423(3):510-523
Using a rapid pH electrode, measurements were made of the flash-induced proton transport in isolated spinach chloroplasts. To calibrate the system, we assumed that in the presence of ferricyanide and in steady-state flashing light, each flash liberates from water one proton per reaction chain. We concluded that with both ferricyanide and methylviologen as acceptors two protons per electron are translocated by the electron transport chain connecting Photosystem II and I. With methyl viologen but not with ferricyanide as an acceptor, two additional protons per electron are taken up due to Photosystem I activity. One of these latter protons is translocated to the inside of the thylakoid while the other is taken up in H2O2 formation. Assuming that the proton released during water splitting remains inside the thylakoid, we compute H+e? ratios of 3 and 4 for ferricyanide and methyl viologen, respectively.In continuous light of low intensity, we obtained the same H+e? ratios. However, with higher intensities where electron transport becomes rate limited by the internal pH, the H+e? ratio approached 2 as a limit for both acceptors.A working model is presented which includes two sites of proton translocation, one between the photoacts, the other connected to Photosystem I, each of which translocates two protons per electron. Each site presents a ≈ 30 ms diffusion barrier to proton passage which can be lowered by uncouplers to 6–10 ms.  相似文献   

2.
Hendrik Hüdig  Gerhart Drews 《BBA》1984,765(2):171-177
Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 H+e? with uptake of 1 H+e? from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K+ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c2-oxidoreductase showed a pumping activity of 0.9 H+e?, which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.  相似文献   

3.
Electron transport, phosphorylation and internal proton concentration were measured in illuminated spinach chloroplast thylakoid membranes under a number of conditions. Regardless of the procedure used to vary these parameters, the data fit a simple chemiosmotic model. Protons from Photosystem II did not appear to be utilized differently from those derived from Photosystem I. The maximal phosphorylation efficiency (Pe2) for photophosphorylation in washed thylakoids under oxidizing conditions is likely to be 43. This value is consistent with a proton-to-electron-pair ratio of 4 for electron flow through both photosystems and a proton-to-ATP ratio of 3 for the chloroplast proton-ATPase.  相似文献   

4.
The proton efflux from intact, anaerobic Escherichiacoli cells following a small oxygen pulse is both slow (t1M2~-10s) and inefficient (H+O~-0.5. Very low levels (<80 nM) of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which have no detectable effect upon active transport, cause a 3–5 fold stimulation in the extent of proton efflux without affecting the efflux rate. At slightly higher concentrations of FCCP (80 nM to 0.5 μM), a sharp inhibition of this increased proton efflux occurs, with the H+O ratio obtained in the presence of 0.5 μM FCCP approximately equal to that obtained in the absence of FCCP. Still higher concentrations of FCCP (> 1 μM), which inhibit active transport, cause a further gradual decrease in the H+O ratio. The unusual increase in the apparent efficiency of H+ efflux by <80 nM FCCP is not accompanied by an increase in the rate of membrane deenergization following an O2 pulse, although such an increase is seen with the higher (uncoupling) FCCP concentrations.  相似文献   

5.
Chloroplasts which were rapidly isolated from illuminated leaves showed activity of ATP hydrolysis at a level much higher than that of the dark control. Under the high-intensity illumination or under repetitive flash excitation, the activated chloroplasts synthesized more ATP than those with a low ATP hydrolysis activity. Δ\?gmH+ formed under repetitive flashes was smaller in the activated chloroplasts than in the inactive chloroplasts. The inhibition of ATP yield per flash by valinomycin or nigericin in the presence of K+ was stronger in the inactive chloroplasts than in the activated chloroplast. ATP synthesis in the activated chloroplasts seems to have a lower Δ\?gmH+ threshold.  相似文献   

6.
A non-alkalophilic mutant strain of Bacillusalcalophilus grows on L-malate over a pH range from 5.0 to 9.0. The mutant does not exhibit the energy-dependent efflux of Na+ that has been used to assay a Na+H+ antiporter in the wild type organism. The mutant also fails to transport α-aminoisobutyric acid, at pH 9.0, either in the presence or absence of Na+; at pH 5.5, the amino acid analogue is taken up by a Na+-independent mechanism. The properties of the mutant constitute strong evidence that the Na+H+ antiporter is involved in maintaining an acidified cytoplasm in B. alcalophilus.  相似文献   

7.
8.
The decarboxylation of pyruvic acid by the thiamine pyrophosphate dependent pyruvate decarboxylase from brewer's yeast is accompanied by a carboxyl carbon isotope effect k12k13 = 1.0083±0.0003 at 25°, pH 6.8. The small size of the isotope effect indicates that decarboxylation is not rate-determining in the overall reaction. The rate constant for decarboxylation of the enzyme-bound pyruvate-thiamine pyrophosphate complex is greater by about a factor of five than the rate constant for dissociation of this complex to form free pyruvate and the enzyme-thiamine pyrophosphate complex.  相似文献   

9.
Diffusion of histamine, theophylline and tryptamine through planar lipid bilayer membranes was studied as a function of pH. Membranes were made of egg phosphatidylcholine plus cholesterol (1 : 1 mol ratio) in tetradecane. Tracer fluxes and electrical conductances were used to estimate the permeabilities to nonionic and ionic species. Only the nonionic forms crossed the membrane at a significant rate. The membrane permeabilities to the nonionic species were: histamine, 3.5 · 10?5cm · s?1; theophylline, 2.9 · 10?4cm · s?1; and tryptamine, 1.8 · 10?1cm · s?1. Chemical reactions in the unstirred layers are important in the transport of tryptamine and theophylline, but not histamine. For example, as pH decreased from 10.0 to 7.5 the ratio of nonionic (B) to ionic (BH+) tryptamine decreased by 300-fold, but the total tryptamine permeability decreased only 3-fold. The relative insensitivity of the total tryptamine permeability to the ratio, [B]/[BH+], is due to the rapid interconversion of B and BH+ in the instirred layers. Our model describing diffusion and reaction in the unstirred layers can explain some ‘anomalous’ relationships between pH and weak acid/base transport through lipid bilayer and biological membranes.  相似文献   

10.
The uptake of radiolabeled carnitine and butyrobetaine has been studied in human heart cells (CCL 27). The uptake of carnitine is 3–10-fold higher in heart cells than in fibroblasts (pmol · μg DNA?1). The uptake of carnitine increases with temperature coefficient KT of 1.6 in the interval 10–20° C and with a negligible uptake at 4 and 10° C. The uptake of carnitine follows Michaelis-Menten kinetics with a KM of 4.8 ± 2.2 μM and V = 8.7 ± 3.2 pmol · μg DNA?1 · h?1. Carnitine uptake is suppressed 90% by NaF (24 mM). Butyrobetaine is taken up into heart cells to the same extent as carnitine with a KM of 5.7–17.3 μM and V = 8.7–9.3 pmol · μg DNA?1 · h?1. Butyrobetaine inhibits competitively the uptake of carnitine and carnitine inhibits the uptake of butyrobetaine to the same extent. No conversion of radiolabeled butyrobetaine to carnitine, or carnitine to methyl choline was observed intra- or extracellulary during incubation. These data are compatible with a selective transport mechanism for carnitine which is also responsible for the uptake of butyrobetaine.  相似文献   

11.
The widely held view that stimulated phagocytes liberate O2? into the extracellular medium is supported by the alterations in oxygen uptake which occur when ferricytochrome c is added to a suspension of zymosan-treated neutrophils. An explanation consistent with this view is provided for some previously reported results (FEBS Lett. 100, 27) which initially appeared to conflict with the notion that O2? is released by phagocytes.  相似文献   

12.
The extended Huckel molecular orbital method was used to predict the preferred conformation of thyrotropin releasing hormone (TRH). Calculation of the whole molecule confirmed the conformational preferences of the individual amino acid residues calculated separately. A stereochemical basis is predicted for the differing activity of N1im methyl and N3im methyl TRH analogues.  相似文献   

13.
The phoS periplasmic protein, implicated in alkaline phosphatase regulation, is shown to be involved in inorganic phosphate (Pi) transport in E. coli. Although phoS? cells dependent upon the PST system for Pi transport can grow in minimal medium with 1 mM Pi as source of phosphorus, the affinity of these cells for Pi is greatly reduced; Km = 18 μM compared with Km = 0.4 μM for phoS+ cells. phoS? cells dependent upon the PST Pi transport system acquire the ability to accumulate Asi from the medium in contrast to phoS+ cells which exclude this toxic anion. It would appear that the periplasmic phoS protein is not essential for Pi accumulation but is involved in maintaining the specificity of the PST Pi transport system.  相似文献   

14.
Leakage of the entrapped anionic fluorophore carboxyfluorescein was used as a measure of the permeability of liposomes to several different acids. Carboxyfluorescein leakage increased with increasing buffer concentration at a given pH and depended on its chemical nature: apolar weak acids such as acetic or pyruvic acids induced fast leakage at relatively high pH (4 to 5), while glycine, aspartic, citric and hydrochloric acids induced leakage only at lower pH. Fluorescence leakage measurements reflected the acidification of the liposomes' aqueous spaces, which was primarily caused by the diffusion of undissociated acid molecules across the lipid bilayer. A simple mathematical model in accord with this hypothesis and assuming that carboxyfluorescein leakage was directly related to the proportion of its neutral lactone form, described satisfactorily the carboxyfluorescein leakage kinetics and allowed rough estimation of permeability coefficients for carboxyfluorescein (neutral lactone form; 9 · 10?9 cm · s?1), acetic acid (>1 · 10?7cm · s?1) and glycine (cation: 6 · 10?9 cm · s?1). These results are consistent with low effective proton permeability of liposomes (<5 · 10?12cm · s?1) and with the permeability coefficient of HCl (3 · 10?3 cm · s?1) reported by Nozaki and Tanford ((1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4324–4328). Diffusion of weak acid molecules across lipid membranes has implications for drug encapsulation and delivery, and may be of biological significance.  相似文献   

15.
Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pKa's are ΔH0 = 8.4 ± 0.3 kcal mol?1, ΔS0 = ?28. ± 1 cal mol?10K?1 for 6-Cy and ΔH0 = 10.0 ± 0.1 kcal mol?1, ΔS0 = ?22.4 ±0.3 cal mol?10K?1 for 7-Cy. Intrinsic 13C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D2O (vv). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 20-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.  相似文献   

16.
The isoelectric points of the blood group A1, A2 and B gene-associated glycosyltransferases in human ovarian cyst fluids were found by isoelectric focusing to be in the pH range 9.5–10. The A1 and B transferases in serum had isoelectric points similar to those of the enzymes in cyst fluids but A2 transferases in serum had considerably lower isoelectric points, in the pH range 6–7. The difference in the pI values of the A1 and A2 transferases in the serum of a donor of the genotype A1A2 enabled the two enzymes to be preparatively separated by the isoelectric focusing technique. The dissimilarity in the pI values of the A2 transferases in ovarian cyst fluids and serum samples indicates that the isoelectric point arises from a post-translational modification of the enzyme protein.  相似文献   

17.
Two membrane fractions prepared from the Ehrlich ascites-tumor cell show non-identical stimulatory responses to certain amino acids in their Mg2+-dependent activity to cleave ATP, despite the presence of ouabain and the absence of Na+ or K+. The first of these, previously described, shows little (Na+ + K+)-ATPase activity, and is characteristically stimulated by the presence of certain diamino acids with low pK2, and at pH values suggesting that the cationic forms of these amino acids are effective. The evidence indicates that these effects are not obtained through occupation of the kinetically discernible receptor site serving characteristically for the uphill transport of these amino acids into the Ehrlich cell. The second membrane preparation was purified with the goal of concentrating the (Na+ + K+)-ATPase activity. It also is stimulated by the model diamino acid, 4-amino-1-methylpiperidine-4-carboxylic acid, and several ordinary amino acids. The diamino acids were most effective at pH values where the neutral zwitterionic forms might be responsible. Among the optically active amino acids tested, the effects of ornithine and leucine were substantially stronger for the l than for d isomers. The list of stimulatory amino acids again corresponds poorly to any single transport system, although the possibility was not excluded that stimulation might occur for both preparations by occupation of a membrane site which ordinarily is kinetically silent in the transport sequence. The high sensitivity to deoxycholate and to dicyclohexylcarbodiimide of the hydrolytic activity produced by the presence of l-ornithine and 4-amino-1-methyl-piperidine-4-carboxylic acid suggests that the stimulatory effect is not merely a general intensification of the background Mg+-dependent hydrolytic activity.  相似文献   

18.
Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca2+ transport in this preparation were characterized and compared to those of (Ca2+ + Mg2+)-ATPase activity. The time course of Ca2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca2+/mg protein after 3–4 min of incubation. The rate of Ca2+ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca2+/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent Km values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent Km values for Mg2+ and Ca2+ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal (Ca2+ + Mg2+)-ATPase. The presence of potassium was required for maximum Ca2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca2+ transport and (Ca2+ + Mg2+-ATPase activity was K+ >Na+ = NH4+ >Li+ . Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM p-chloromercuribenzene sulfonate. The results indicate that Ca2+ transport in adipocyte endoplasmic reticulum is mediated by a (Ca2+ + Mg2+)-ATPase and suggest an important role for endoplasmic reticulum in control of intracellular Ca2+ distribution.  相似文献   

19.
Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation kn = ko (1 ? n + nπa1)4 with πa1 ~ 0.74, where ko is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, kn is the rate constant in a protium oxide-deuterium oxide mixture, and πa1 is the isotopic fractionation factor.  相似文献   

20.
A method for calculating the rate constant (KA1A2) for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of KA1A2 obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: (4.5±1.4) · 103s?1 and (6.9±1.2) · 103 s?1, respectively.The proposed method has also been used for the estimation of the KA1A2 value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of KA1A2 has been found to be (2.7±0.8) · 103 s?1.The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.  相似文献   

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