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At least 19 recordsLinked to original sources
PubMed1976

Different proton-sugar stoichiometries for the uptake of glucose analogues by Chlorella vulgaris. Evidence for sugar-dependent proton uptake without concomitant sugar uptake by the proton-sugar symport system.

The uptake of hexoses by Chlorella vulgaris is accompanied by the uptake of protons. For 6-deoxyglucose a stoichiometry of one proton taken up per sugar molecule has been measured, whereas for 1-deoxyglucose approximately two protons are taken up per sugar molecule. It was found that in the presence of 1-deoxyglucose a considerable proportion of "carrier" catalyzes the transport of protons without the concomitant transport of sugar. Presumably, the binding of sugar initiates the translocation of the carrier-proton-sugar complex, but whereas 1-deoxyglucose can still dissociate from the complex at the external side of the cytoplasmic membrane, the translocation of the carrier-proton complex continues. This conclusion was reached since (a) the composition of the translocated carrier-proton-sugar complex is the same for both sugar. Its formation is a first order reaction with respect to protons. (b) When 6-deoxyglucose, present inside cells, is exchanged for external sugar, the exchange ratio is two to one when the external sugar is 1-deoxyglucose, two molecules of 6-deoxyglucose are lost for each molecule of 1-deoxyglucose entering. This result indicates that during uptake of 1-deoxyglucose statistically only each second carrier molecule appearing at the internal side of the cytoplasmic membrane is carrying sugar.

Binding Sites↗
PubMed1977

Purified proton conductor in proton translocating adenosine triphosphatase of a thermophilic bacterium.

1. The membrane-integrated portion (TF0) of the proton translocating ATPase complex (TF0-F1) of the thermophilic bacterium PS3 was highly purified. Its proton-conducting activity was investigated in vesicles reconstituted from TF0 and phospholipids (TF0 vesicles). 2. The rate of proton conduction through TF0 was proportional to the membrane potential imposed (6H+ uptake/s/TF0 molecule with 103 mV at pH 8.0). The pH profile of the rate revealed that a proton, not a hydroxy ion, was the true substrate conducted and that there was a monoprotic proton binding site in TF0 (pKa = 6.8). The temperature coefficient of proton conductance of TF0 showed a considerable variation depending on the phospholipids of the vesicles with respective transition temperatures. 3. Passive proton conduction through TF0 was inhibited stoichiometrically by addition of either the soluble ATPase portion (TF1) of TF0-F1, or an energy transfer inhibitor dicyclohexylcarbodiimide or an antibody against TF0. 4. The proton conductance of TF0 was concluded to represent its intrinsic activity in the original TF0-F1 complex.

Adenosine Triphosphatases↗
PubMed1977

The effective proton conductance of the inner membrane of mitochondria from brown adipose tissue. Dependency on proton electrochemical potential gradient.

The nucleotide-sensitive H+ (OH-) conducting pathway of mitochondria from the brown-adipose tissue of cold-adapted guinea-pigs passes an effective proton current which is directly proportional to the proton electrochemical gradient. At 23 degrees C and pH 7.0 this conductance is 16 nmol H+ - min-1 - mg-1 - mV-1. Addition of 0.2 mM GDP results in a conductance which is linear and low (0.7 nmol H+ - min-1 - mg-1 - mV-1) until deltamicronH+ exceeds 220 mV. At higher values of deltamicronH+, which can be attained by glycerol 3-phosphate oxidation but not palmitoyl-L-carnitine plus malate oxidation, the membrane conductance greatly increases, effectively limiting the maximal deltamicronH+ to 240 mV. High glycerol 3-phosphate concentrations which have the thermodynamic potential to exceed this value of deltamicronH+ instead create a greatly increased rate of controlled respiration. The generality and significance of this device to limit deltamicronH+, and its relation to the nucleotide-sensitive conductance, are discussed.

Acclimatization↗
PubMed1976

Electrochemical potential of protons in vesicles reconstituted from purified, proton-translocating adenosine triphosphatase.

Measurements were made of the difference in the electrochemical potential of protons (delta-mu H+) across the membrane of vesicles restituted from the ATPase complex (TF0.F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (delta psi) and pH difference across the membrane (delta pH), respectively. In the presence of Tris buffer the maximal delta psi ans no delta pH were produced, while in the presence of the permeant anion NO-3 the maximal delta pH and a low delta psi were produced by the addition of ATP. When thATP concentration was 0.24 mm, the delta psi was 140-150 mV (positive inside) in Tris buffer, and the delta pH was 2.9-3.5 units (acidic inside) in the presence of NO-3. Addition of a saturating amount of ATP produced somewhat larger delta psi and delta pH values, and the delta -muH+attained was about 310mV. By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4-5 during ATP hydrolysis. The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.

Adenosine Triphosphatases↗
PubMed1979

Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase. Assignment of histidine C-2 protons in binary complexes with folates on the basis of the crystal structure with methotrexate and on chemical modifications.

The effects of pH upon the C-2 resonances of the 5 histidine residues of Escherichia coli MB 1428 dihydrofolate reductase in binary complexes with methotrexate, aminopterin, folate, methopterin, and trimethoprim were studied by 300-MHz 1H nmr spectroscopy. Three of the five histidine residues, labeled 1, 2, and 3, exhibited similar pK' values and chemical shifts for their C-2 protons in the five binary complexes. One histidine, 4, was quite different in the folate complex and the last histidine, 5 was quite different in the trimethoprim complex. For all five binary complexes, each histidine had a pK' which was significantly different from the other 4 histidines of that complex. Titration of the binary methotrexate complex of a 5,5'-dithiobis(2-nitrobenzoate)-modified enzyme showed that 2 histidines were not perturbed by this modification of Cys 152, and that the alkaline form of histidine 2, the acid form of histidine 4, and, to a lesser extent, the acid form of histidine 3 were slightly perturbed. Titration of the binary methotrexate complex of a N-bromosuccinimide-modified enzyme demonstrated that this modification slightly affected all of the histidines and drastically affected histidine 5. Histidines 3 and 5 of the binary methotrexate complex reacted rapidly with the histidine-specific reagent, ethoxyformic anhydride, while histidines 2 and 4 reacted at a moderate rate and histidine 1 reacted slowly if at all. The local electrostatic environments of the 5 histidine residues as deduced from the crystal structure of the binary complex of the enzyme with methotrexate (Matthews, D.A., Alden, R.A., Bolin, J.T., Freer, S.T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M.N., and Hoogsteen, K. (1977) Science 197, 594-597) were used as the basis for proposed assignments of the five histidine C-2 nmr resonances. The assignments were: 1, pK' 7.9 to 8.2, His 124; 2, pK' 7.2 to 7.4, His 141; 3, pK' 6.5 to 6.7, His 149; 4, pK' 5.7 to 6.3, His 114; and 5, pK' 5.2 to 5.9, His 45. The effect of the chemical modifications upon the enzyme's histidine residues were consistent with the assignments, but no direct chemical evidence in support of the assignments was obtained. It was proposed that, since the crystallographic data provided consistent assignments of the histidine nmr data for both native and chemically modified enzyme, the local environment of each of the 5 histidine residues was similar in the crystal and in solution.

Binding Sites↗
PubMed1977

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.

Adenosine Diphosphate↗
PubMed1976

Relations between the electrical potential, pH gradient, proton flux and phosphorylation in the photosynthetic membrane.

The transmembrane electrical potential (deltaphi), the proton flux (H+), the rate of electron transport (e), the pH gradient (deltapH) and the rate of phosphorylation (ATP) were measured in chloroplasts of spinach. Photosynthesis was excited periodically with flashes of variable frequencies and intensities. A new method is described for determining the rate of electron transport and proton flux. Under conditions where the rate of electron transport and proton flux are not pH controlled the following correlations were found in the range 50 mV less than or equal to deltaphi less than or equal to 125 mV and 1.8 less than or equal to deltapH less than or equal to 2.7: (1) The pH gradient, deltapH, increases with H+ independently of Phout between 7-9. (2) The rate of phosphorylation, ATP, depends exponentially on deltapH (at constant deltaphi) and is independent of pHout between 7-9. (3) The rate of phosphorylation, ATP, depends also on deltaphi (at constant deltapH and at constant proton flux H+). (4) The proton flux via the ATPase pathway, Hp+, depends non-linearly on the ratio of the proton concentrations: Hp+ approximately (Hin+/Hout+)b, (b=2.3--2.6). The proton flux via the basal pathway, Hb+, depends linearly on the ratio of the proton concentrations: Hb+ approximately (Hin/Hout). (5) The ratio deltaH+/ATP (e/ATP, i.e. the ratio of the total proton flux, Hp+ + Hb+, and the rate of ATP formation, ATP, depends strongly on deltaphi and on deltapH. The ratio is deltaH+/ATP approximately 3 (e/ATP approximately 1.5) at deltapH 2.7 and deltaphi = 125 mV. (6) It is supposed that the reason for the dependence of deltaH+/ATP on deltaphi anddeltapH is the different functional dependence of the basal proton flux Hb+ and the phosphorylating proton flux Hp+ on deltapH and deltaphi. The calculation of deltaH+/ATP on the basis of this assumption is in fair agreement with the experimental values. Also the "threshold" effects can be explained in this way. (7) The ratio of deltaHp+/ATP, i.e. the ratio of the phosphorylating proton flux Hp+ and ATP, is deltaHp+/ATP APPROXIMATELY 2.4.

Adenosine Triphosphate↗
PubMed1978

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.

Cyclic GMP↗
PubMed1977

Gated proton conduction via the coupling factor of photophosphorylation modified by N,-N-orthophenyldimaleimide.

The membrane bound coupling factor of photophosphorylation is studied after pretreatment of broken chloroplasts with the bifunctional N,N-orthophenyldimaleimide under energization of the thylakoid membrane by mild flashing light. The proton conduction of the membrane is monitored both via the electrochromic absorption changes and via selective pH-indicating dyes. It is found that the coupling factor, after interaction with N,N-orthophenyldimaleimide during the preillumination period, shortcircuits one of the two protons pumped inside after excitation of chloroplasts with one short flash of light. In contrast to the low proton conductivity of the unperturbed thylakoid membrane (relaxation time for a proton gradient greater than 5s), this extra proton channel leads to a partial relaxation of a proton gradient within a few ms. Although limited to only one proton per electron, this extra proton conducting pathway is not otherwise specific. It operates with protons resulting from both Photosystem I and Photosystem II activity. In addition it operates with protons already present in the internal phase before firing of the exciting light flash. These effects are prevented by the presence of ATP (but not GTP) during the preillumination period. It is suggested that the modified coupling factor is gated open by the light induced electric field across the thylakoid membrane while self closing after passage of one proton per activated coupling factor.

Chemical Phenomena↗
PubMed1978

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles.

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated. Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H+ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K+. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons. The proton ejection is the result of true translocation of H+ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system. Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a H+/e- quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions. It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H+.

Aerobiosis↗
PubMed1976

A simple, quantitative approach to the coupling of photophosphorylation to electron flow in terms of proton fluxes.

A simple relationship between observed phosphorylation efficiencies (P/e ratios) and internal proton concentration in spinach chloroplast thylakoids has been derived. P/e ratios, varked by either changing the light intensity or by adding the energy transfer inhibitor, 4'-deoxyphlorizin, were found to change with internal proton concentration in accordance with this relationship. A quantitative prediction of the effect of uncouplers on the P/e ratio can probably also be made. By extrapolation of plots of observed P/e ratios against internal proton concentration divided by the overall rate of electron flow, a maximum intrinsic P/e of about 0.66 is obtained. Assuming that two protons appear inside thylakoids per electron transferred, a P/e ratio of 0.66 suggests that three internal protons are consumed for each ATP formed. Internal protons may be considered to be substrates for the phosphorylation reaction. Hill plots of phosphorylation rate vs. internal proton concentration also indicate that three protons are consumed for each ATP synthesized. Thus, the H+ concentration gradient behaves quantitatively, as well as qualitatively, as if it is the connecting link between electron flow and phosphorylation in illuminated thylakoids.

Chloroplasts↗
PubMed1979

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential gradient, and that the maximal rate of accumulation may exist when both components of the protonmotive force are present.

Adenosine Triphosphate↗
PubMed1976

Light-driven proton translocations in Halobacterium halobium.

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.

Bacteriorhodopsins↗
PubMed1979

Studies on the proton transport at system II in trypsin-treated spinach chloroplasts.

The proton transport coupled with the DCMU-insensitive oxygen evolution mediated by K3[Fe(CN)6] in trypsin-treated chloroplasts (Renger, G. (1976) FEBS Lett. 69, 225--230) has been investigated with the aid of the pH indicator bromcresol purple. It was found that (1) the proton uptake from the outer aqueous phase observed in normal chloroplasts is completely suppressed by mild trypsin treatment; (2) a rather slow proton release into the external phase is detected which is insensitive to DCMU; (3) in the presence of DCMU, the extent of the proton release depends on the incubation time with trypsin in a similar manner as the average oxygen yield per flash. The results are interpreted by the assumption, that: (i) the reduced primary electron acceptor of System II, X 320-, does not become protonated, and (ii) the external acidification is caused by a passive efflux of protons, which are released by the watersplitting enzyme system Y into the inner phase of the thylakoids. The pK value of X 320- in trypsinated chloroplasts is estimated to be below 4.5. A possible pK shift caused by a modification of the proteinaceous barrier, which earlier (Renger, G. (1976) Biochim. Biophys. Acta 440, 287--300) was postulated to cover up the primary electron acceptor X 320, is discussed. Furthermore, the watersplitting enzyme system Y is inferred to be sensitive to deletereous attack from the outer aqueous phase mainly by secondary structural effects. Trypsination does not change the direction of the proton release in system Y.

Chloroplasts↗
PubMed1975

Succinate uptake and related proton movements in Escherichia coli K12.

1. The apparent Km values for succinate uptake by whole cells of Escherichia coli K12 depend on pH in the range 6.5-7.4.2. Uptake of succinate in lightly buffered medium is accompanied by proton uptake. 3. The apparent Km values for succinate uptake and for succinate-induced proton uptake are similar. 4. Approximately two protons enter the cell with each succinate molecule. 5. The pattern of inhibition of succinate uptake is similar to that of succinate-induced proton uptake. 6. Uptake of fumarate and malate, which share the succinate-transport system, is also accompanied by the uptake of approximately two protons per molecule of fumarate or malate. 7. Uptake of aspartate by the dicarboxylic acid-transport system is accompanied by the uptake of approximatley two protons per molecule of asparatate. 8. It is concluded that uptake of dicarboxylic acids by the dicarboxylic acid-transport system is obligatorily coupled to proton uptake such that succinate, malate and fumarate are taken up in electroneutral form and asparate is taken up in cationic form. 9. These results are consistent with, though they do not definitely prove, the energization of succinate uptake of the deltapH.

Aerobiosis↗
PubMed1978

The microbial metabolism of Cl compounds. The stoicheiometry of respiration-driven proton translocation in Pseudomonas AM1 and in a mutant lacking cytochrome c.

This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.

Cyanides↗