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K J Hellingwerf

Publications and source records attributed to K J Hellingwerf.

At least 91 records · Page 5Linked to original sources
PubMed1989

On the evaluation of data from flow-dialysis experiments.

Flow dialysis can be used to measure (i) ligand binding to macromolecules and (ii) the size of transmembrane ion gradients. Generally an approximate method is used to calculate the binding or gradient parameters from the raw data. Here we present a simple but exact method and evaluate the errors that may arise when the approximate method is used to calculate the magnitude of ion gradients. In addition, equations are presented that allow for a correction for sampling from or additions to the upper compartment of a flow-dialysis vessel during the measurements. Setty and Hendler [(1982) J. Biochem. Biophys. Methods 7, 35-46] have reported artifacts in the measurement of ion-gradients caused by the addition of electron donors to the upper compartment of a flow-dialysis cell. Here we extend their observations and suggest additional methods to prevent such artifacts.

Biopolymers↗
PubMed1989

Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH.

The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W. N. Konings, J. Bacteriol. 170:5647-5653, 1988). The dependence of solute transport in R. sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system.

Alanine↗
PubMed1988

Effects of potassium ions on proton motive force in Rhodobacter sphaeroides.

The proton motive force (PMF) was determined in Rhodobacter sphaeroides under anaerobic conditions in the dark and under aerobic-dark and anaerobic-light conditions. Anaerobically in the dark in potassium phosphate buffer, the PMF at pH 6 was -20 mV and was composed of an electrical potential (delta psi) only. At pH 7.9 the PMF was composed of a high delta psi of -98 mV and was partially compensated by a reversed pH gradient (delta pH) of +37 mV. ATPase inhibitors did not affect the delta psi, which was most likely the result of a K+ diffusion potential. Under energized conditions in the presence of K+ the delta psi depolarized due to electrogenic K+ uptake. This led to the generation of a delta pH (inside alkaline) in the external pH range of 6 to 8. This delta pH was dependent on the K+ concentration and was maximal at external K+ concentrations larger than 1.2 mM. In energized cells in 50 mM KPi buffer containing 5 mM MgSO4, a delta pH (inside alkaline) was present at external pHs from pH 6 to 8. As a result the overall magnitude of the PMF at various external pHs remained constant at -130 mV, which was significantly higher than the PMF under anaerobic-dark conditions. In the absence of K+, in 50 mM NaPi buffer containing 5 mM MgSO4, no depolarization of the delta psi was found and the PMF was composed of a large delta psi and a small delta pH. The delta pH became even reversed (inside acidic) at alkaline pHs (pH>7.3), resulting in a lowering of the PMF. These results demonstrate that in R. sphaeroides K+ uptake is essential for the generation of a delta pH and plays a central role in the regulation of the internal pH.

Aerobiosis↗
PubMed1988

Light-driven amino acid uptake in Streptococcus cremoris or Clostridium acetobutylicum membrane vesicles fused with liposomes containing bacterial reaction centers.

Reaction centers of the phototrophic bacterium Rhodopseudomonas palustris were introduced as proton motive force-generating systems in membrane vesicles of two anaerobic bacteria. Liposomes containing reaction center-light-harvesting complex I pigment protein complexes were fused with membrane vesicles of Streptococcus cremoris or Clostridium acetobutylicum by freeze-thawing and sonication. Illumination of these fused membranes resulted in the generation of a proton motive force of approximately -110 mV. The magnitude of the proton motive force in these membranes could be varied by changing the light intensity. As a result of this proton motive force, amino acid transport into the fused membranes could be observed. The initial rate of leucine transport by membrane vesicles of S. cremoris increased exponentially with the proton motive force. An H+/leucine stoichiometry of 0.8 was determined from the steady-state level of leucine accumulation and the proton motive force, and this stoichiometry was found to be independent of the magnitude of the proton motive force. These results indicate that the introduction of bacterial reaction centers in membrane vesicles by the fusion procedure yields very attractive model systems for the study of proton motive force-consuming processes in membrane vesicles of (strict) anaerobic bacteria.

Amino Acids↗
PubMed1987

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris.

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.

Amino Acid Transport Systems↗
PubMed1987

Reaction centers from Rhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. I. Structural studies.

Reaction centers (RCs) from Rhodopseudomonas sphaeroides were reconstituted into asolectin vesicles by cosonication. Equilibrium centrifugation on sucrose gradients showed that the vesicles were homogeneous in density (i.e., lipid-to-protein ratio) when reconstituted at a molar lipid-to-protein ratio between 500 to 1000. At lower ratios, a considerable fraction of RCs was not incorporated into closed vesicles, while at higher ratios, an increasing population of liposomes was protein-free. The average vesicle size decreased with increasing lipid-to-protein ratio, exhibiting considerable size heterogeneity within a sample. The average diameter of the largest and smallest population of vesicles, reconstituted at a molar lipid-to-protein ratio of 560, was 1200 and 400 nm, respectively. The orientation of reconstituted RCs with respect to the plane of the membrane was determined from the flash-induced rereduction kinetics of the special-pair bacteriochlorophyll dimer in the presence of reduced cytochrome c. The predominant orientation of RCs was such that the cytochrome c binding sites faced the external medium. The net orientation of RCs in reconstituted vesicles decreased with vesicle size and was strongly influenced by the ionic strength during reconstitution.

Bacterial Proteins↗
PubMed1987

Reaction centers from Rhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation.

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochrome c and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ0), a lipid- and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ0H2) and oxidized cytochrome c. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochrome c and UQ0. At 250 and 25 microM UQ0 and cytochrome c, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.

Bacterial Proteins↗
PubMed1987

Regulation of the glutamate-glutamine transport system by intracellular pH in Streptococcus lactis.

Various methods of manipulation of the intracellular pH in Streptococcus lactis result in a unique relationship between the rate of glutamate and glutamine transport and the cytoplasmic pH. The initial rate of glutamate uptake by S. lactis cells increases more than 30-fold when the intracellular pH is raised from 6.0 to 7.4. A further increase of the cytoplasmic pH to 8.0 was without effect on transport. The different levels of inhibition of glutamate and glutamine transport at various external pH values by uncouplers and ionophores, which dissipate the proton motive force, can be explained by the effects exerted on the intracellular pH. The dependence of glutamate transport on the accumulation of potassium ions in potassium-filled and -depleted cells is caused by the regulation of intracellular pH by potassium movement.

Biological Transport, Active↗
PubMed1987

Membrane systems in which foreign proton pumps are incorporated.

Detailed information about the role of the proton-motive force in solute transport has often been obtained from studies in model systems such as membrane vesicles. For many bacteria, such studies have been hampered by the lack of a good proton-motive force generating system in these model systems. Recently this problem has been solved by the development of procedures to incorporate foreign proton pumps in membrane vesicles derived from bacteria. This improved model system has been used for studies on the role of the proton-motive force in solute transport in fermentative bacteria and yeasts. Important applications can be found in studies of many energy-transducing systems in membranes which lack a suitable proton pump.

Bacteria↗
PubMed1986

Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles.

Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system.

Bacillus subtilis↗
PubMed1985

The role of the proton motive force and electron flow in solute transport in Escherichia coli.

Transport of lactose and methyl beta-D-thiogalactopyranoside, a melibiose analogue, was studied in intact cells of Escherichia coli. A proton motive force could drive the translocation of these solutes via these two transport systems, but the initial rates and steady-state levels of solute accumulation increased upon initiation of electron transfer. When the absolute value of the proton motive force was decreased by ionophores the steady-state levels of lactose accumulation did not decrease as expected if thermodynamic equilibrium with the proton motive force had existed. Accumulation of lactose was also observed in the absence of any measurable proton motive force as long as electron transfer took place. Since both proton/lactose and sodium/methyl beta-D-thiogalactopyranoside symport showed the same characteristics, an explanation based on local proton diffusion pathways is unlikely.

Anaerobiosis↗
PubMed1985

A polyvinylchloride-membrane based anion selective electrode for continuous registration of delta pH (interior alkaline) with salicylate as the indicator probe.

An anion sensitive electrode has been constructed with the use of the lipid soluble cation benzyl-dimethyl-hexadecylammonium analogous to the procedure described for tetraphenylphosphonium-sensitive electrodes [Shinbo, T., Kamo, N., Kurihara, K. and Kobatake, Y. (1978) Arch. Biochem. Biophys. 187, 414-422]. The anion electrode responds to salicylate concentrations above 400 microM with a Nernstian sensitivity. Less lipid soluble anions like chloride and phosphate do not interfere. Below 400 microM salicylate the response of the electrode decreases gradually so that the sensitivity of the electrode is less than 10 mV per decade change at concentrations of the anion of 50 microM. A computer program has been developed to fit the electrode response curve with a polynomal function of the fourth power. Additional software-allows calculation of changes in the concentration of the salicylate anion, also under conditions where the sensitivity of the electrode for the anion is not constant. In this way the electrode can be used to measure changes in salicylate concentration that occur in a suspension of bacteria when, upon energization, a pH gradient is generated. 31P nuclear magnetic resonance measurements demonstrated that the pH gradient measured with the salicylate-sensitive electrode in the phototrophic bacterium Rhodopseudomonas sphaeroides is quantitatively correct. The response time of the electrode decreases from 1 min at 20 microM salicylate to 10 s at concentrations greater than or equal to 200 microM.

Electrodes↗
PubMed1985

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.

Acinetobacter↗
PubMed1984

The protein composition of the cytoplasmic membrane of aerobically and anaerobically grown Escherichia coli.

The protein composition of the cytoplasmic membranes of Escherichia coli, grown aerobically and anaerobically on a glucose minimal medium at pH 7.0, were analyzed by crossed immunoelectrophoresis. Qualitative differences are limited to only two proteins: nitrate reductase (E.C. 1.7.99.4) is absent under aerobic growth conditions, whereas an unidentified protein, with a molecular weight of 81,500 and located at the inner side of the cytoplasmic membrane, is synthesized only in the presence of oxygen. Quantitative differences are observed for many proteins: the ratio of the amount of a specific protein present in cells grown anaerobically and aerobically was, for four proteins, between 0.3 and 1; for 25 proteins, between 1 and 3; and for five proteins, larger than 5.

Aerobiosis↗
PubMed1984

The interaction between electron transfer, proton motive force and solute transport in bacteria.

The properties of proton solute symport have been studied in Streptococcus cremoris, Rhodopseudomonas sphaeroides and Escherichia coli. In the homolactic fermentative organism S. cremoris the efflux of lactate is a membrane protein-mediated process, which can lead to the generation of a proton motive force. These observations support the energy-recycling model that postulates the generation of metabolic energy by end-product efflux. Studies with oxidants and reductants and specific dithiol reagents in E. coli membrane vesicles demonstrated the presence of two redox-sensitive dithiol-disulphide groups in the transport proteins of proline and lactose. The redox state of these groups is controlled by the redox potential of the environment and by the proton motive force. One redox-sensitive group is located at the inner surface, the other at the outer surface of the membrane. In Rps. sphaeroides and E. coli the activity of several transport proteins depends on the activity of the electron transfer systems. On the basis of these results a redox model for proton solute transport coupled in parallel to the electron transfer system is postulated.

Bacteria↗