共查询到20条相似文献,搜索用时 15 毫秒
1.
Klaus Jann Shiro Kanegasaki Gerd Goldemann P.Helena Mäkelä 《Biochemical and biophysical research communications》1979,86(4):1185-1191
From phosphomannose isomerase-less mutants of strains 08 and 09, derivatives were constructed by recombination with a donor. In contrast to membranes from the parent strains, those from the recombinants did not synthesize the 08 or 09 mannan from GDP mannose . They could, however, be restored to biosynthetic activity with butanol extracts from the bacteria. This indicated that the mutation affects the synthesis of a hydrophobic acceptor. 相似文献
2.
ADP and Pi-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with . ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔGp) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na+ or K+ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low . 相似文献
3.
(1) +/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios , , for reduction to N2 and for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. , for reduction to N2 and for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The ratios, mentioned in item 2, were not altered in the presence of and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The acceptor ratios predicted by this scheme are in good agreement with the experimental values. 相似文献
4.
Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium+ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of . These data cannot be reconciled with a delocalized chemiosmotic coupling model. 相似文献
5.
Mitsumasa Okamoto Hideo Yamagishi 《International journal of biological macromolecules》1981,3(2):105-113
In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA?-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In cells, however, most petit λ was not bound to DNA. In Fec? cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI?-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein pNu1 is required for complex formation between head precursors and DNA concatemers, for the initiation of DNA packaging, and I for the promotion of DNA packaging, and for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques. 相似文献
6.
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 with uptake of 1 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 , 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
7.
The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygenand ferricyanide pulses, with endogenous substrates or added methanol as a substrte, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of . The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans. 相似文献
8.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts Y.-F. Fu G.A.J. Kuijpers J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1984,774(2):277-287
(1) Treatment of from rabbit kidney outer medulla with the labeled thio-analogue of ATP in the presence of and the absence of K+ leads to thiophosphorylation of the enzyme. The value for [γ-S]ATP is 2.2 μM and for Na+ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient . (2) The thio-analogue () can also support overall activity, but at 37°C is only h?1 or 0.09% of the specific activity for ATP (). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s?1 vs. 180 s?1), spontaneous dethiophosphorylation (0.04 s?1 vs. 0.5 s?1) and K+-stimulated dethiophosphorylation (0.54 s?1 vs. 230 s?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s?1, ) and is relatively K+-insensitve. The for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E1-conformation. 相似文献
9.
Furosemide () inhibits a proportion of the total passive (ouabain-insensitive) K+ influx into primary chick heart cell cultures (85%), BC3H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K+ influx is independent of ( inhibition since the furosemide-sensitive component of the K+ influx is identical in the presence and absence of ouabain (). For HeLa cells the passive, furosemide-sensitive component of K+ influx is markedly dependent upon the external K+, Na+ and Cl? content. Acetate, iodide and nitrate are ineffective as substitutes for Cl?, whereas Br? is partially effective. Partial Cl? replacement by NO3? gave an apparent affinity of 100 mM [Cl]. Na+ replacement by choline+ abolishes the furosemide-sensitive component, whereas Li+ replacement reduces this component by 48%. Partial Na+ replacement by choline+ gives an apparent affinity of 25 mM [Na+]. Variation in the external K+ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K+ inflúx is of high affinity, half-maximal inhibition being observed at furosemide. Piretanide () and phloretin () inhibit the same component of passive K+ influx as furosemide; ethacrynic acid and amiloride () partially so. The stilbene, SITS (), was ineffective as an inhibitor of the furosemide-sensitive component. 相似文献
10.
Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells 总被引:2,自引:0,他引:2
M1 cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells () to mature macrophages () within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While cells have no phagocytic activity nor Fc receptor (FcR), cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on cells is resistant to trypsin and pronase. cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. cells lack this macrophage-substituting capacity. Mm1 cells, mutant cells from M1 cells, having FcR and higher phagocytic activity than cells, are also devoid of this capacity. 相似文献
11.
Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of . Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. was found to be closely associated with acylCoA: lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K+ concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by the . 相似文献
12.
13.
A thermodynamic characterization of the Na+-H+ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na+-H+ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, , was in the range of 850–1150 nmol H+·(mg protein)?1·h?1·(pH unit)?1 for four different preparations; for the total Na+ permeability, , it was 1620–2500 nmol Na+·(mg protein)?1·h?1·(pNa unit)?1; and for the proton ‘cross-permeability’, , it was 220–580 nmol H+·(mg protein)?1·h?1·(pNa unit)?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na+-H+ exchange (ηmax), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; ηmax ? 5%; 0.36 ? r ? 2; ?JNa+ ? 1.3 · 105μmol · (RT unit)?1 at JNa = 1 μmolNa+ · (mgprotein)?1 · h?1; and ?ΔpNa ? 5 · 104 ΔpNa · (mg protein) · h · (RT unit)?1 at ΔpNa = 1 unit, for different preparations. 相似文献
14.
Sam W. Woo Anthony W. Linnane Phillip Nagley 《Biochemical and biophysical research communications》1982,109(2):455-462
strain DC5-E2 that lacks mtDNA () can be cotransformed with a mixture of mtDNA and the plasmid YEp13 (LEU2/2μ/pBR322) to produce Leu+ transormants which, on being mated to tester strains, generate respiratory competent diploids (such events are denoted marker rescue). In this work strain DC5-E2 was transformed with recombinant molecules consisting of a mtDNA segment including the gene inserted into YEp13. The Leu+ transformants made with the recombinant plasmids were unable to rescue testers carrying mutations in the region, in contrast to Leu+ cotransformants made with mixtures of YEp13 and mtDNA. We conclude that marker rescue events occur as a result of interactions between the mtDNA of the tester and the mtDNA sequences introduced by transformation. Such interactions cannot occur when the latter mtDNA is forced to replicate in covalent association with YEp13, probably in the nucleus. 相似文献
15.
Françoise Giraud Michel Claret K.Richard Bruckdorfer Bernadette Chailley 《生物化学与生物物理学报:生物膜》1981,647(2):249-258
Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments. 相似文献
16.
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. 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 threshold. 相似文献
17.
W.McD. Armstrong W.R. Bixenman K.F. Frey J.F. Garcia-Diaz M.G. ORegan Jeanie L. Owens 《生物化学与生物物理学报:生物膜》1979,551(1):207-219
Na+, K+ and Cl? concentrations () and activities (), and mucosal membrane potentials () were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl? and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. was measured with conventional open tip microelectrodes, with solid-state Cl?-selective silver microelectrodes and and with Na+- and K+-selective liquid ion-exchanger microelectrodes. The average recorded was ?34 mV. , and were 51, 105 and 52 mM. The corresponding values for , and were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl?. significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl? is implicated in intracellular Cl? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl? electrochemical potential differences (Δμ̄Na and Δμ̄Cl). Δμ̄Na (?7000 J · mol?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ̄Cl (1000–2000 J · mol?1). 相似文献
18.
J K Ladha P Rowell W D Stewart 《Biochemical and biophysical research communications》1978,83(2):688-696
5-hydroxylysine, an analogue of glutamate and lysine, causes production by N2-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of on heterocyst production and C2H2 reduction and in cultures results in heterocyst synthesis and in C2H2 reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary assimilation in A. cylindrica, that alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both. 相似文献
19.
(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献
20.
The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K+ in the presence of valinomycin, and cation influx was coupled to an efflux of H+ using the protonophore . Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be and that to cations . These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed. 相似文献