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PubMed2003

The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA.

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.

ATP-Binding Cassette Transporters↗

PubMed2004

Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel.

Activity of the epithelial Na(+) channel (ENaC) is rate-limiting for Na(+) (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, alpha, beta, and gamma, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2alpha:1beta:1gamma stoichiometry and a higher ordered channel with a 3alpha:3beta:3gamma stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of approximately 90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3alpha:3beta:3gamma stoichiometry.

Animals↗

PubMed1997

Changes in photosystem stoichiometry in response to environmental conditions for cell growth observed with the cyanophyte Synechocystis PCC 6714.

Changes in photosystem stoichiometry in response to shift of environments for cell growth other than light regime were studied with the cyanophyte Synechocystis PCC 6714 in relation to the change induced by light-quality shift. Following two environment-shifts were examined: the shift of molecular form of inorganic carbon source for photosynthesis from CO2 to HCO3- (CO2 stress) and the increase in salinity of the medium with NaCl (0.5 M) (Na+ stress). Both CO2 and Na+ stresses induced the increase in PSI abundance resulting in a higher PSI/PSII stoichiometry. CO2 stress was found to elevate simultaneously Cyt c oxidase activity (Vmax). The feature was the same as that caused by light-quality shift from preferential excitation of PSI to PSII (light stress) though the enhancement by either stress was smaller than that by light stress. Under our experimental conditions, PSI/PSII stoichiometry appeared to increase at a fairly constant rate to the basal level even when the basal level had been differently determined by the light-quality. Enhancing rates for PSI/PSII stoichiometry and for Cyt c oxidase activity were also similar to each other. Since the two stresses affect the thylakoid electron transport similarly to the shift of light-quality, we interpreted our results as follows: three environmental stresses, CO2, Na+, and light stresses, cause changes in electron turnover capacity of PSI and Cyt c oxidase under a similar, probably a common, mechanism for monitoring redox state of thylakoid electron transport system.

Carbon Dioxide↗

PubMed2006

Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment.

The chimeric anti-CD30 IgG1, cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys-->Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.

Animals↗

PubMed1990

Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don.

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H(+) efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H(+)/H(2)PO(4) (-) stoichiometry of the cotransport is most likely to be 4.

Journal Article↗

PubMed1991

Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue.

The H(+)/ATP stoichiometry was determined for the plasma membrane H(+)-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H(+)/ATP stoichiometry utilized a kinetic approach where rates of H(+) influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H(+) influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H(+) leakage from a steadystate pH gradient after stopping the H(+)-ATPase or utilizing a mathematical model which describes the net transport of H(+) at any given point in time. When the rate of H(+) influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H(+)/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (DeltaG(atp)), this value for H(+)/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.

Journal Article↗

PubMed1983

High-field phosphorus NMR studies of the stoichiometry of the lactate/proton carrier in Streptococcus faecalis.

High-field 31P-NMR studies of whole cells of Streptococcus faecalis have shown that delta pH can be formed by ATP hydrolysis and also by lactate transport. We have used 31P-NMR to measure the pH dependence of the variable stoichiometry of the proton/lactate carrier. At low external pH (pH approximately equal to 6.5) the influx stoichiometry was 1.1 H+/lactate, while at high pH (7.5) the ratio was almost 2; the apparent midpoint pH of this variable stoichiometry is 7. delta psi measurements support the electrogenic nature of lactate transport at high pH; the variable rate of membrane depolarization caused by lactate transport also had a midpoint near pH 7.0. The data is consistent with a symmetrical carrier operating with variable stoichiometry as proposed by Michels et al.

Biological Transport↗

PubMed1988

Membrane-potential-dependent changes in the stoichiometry of charge translocation by the mitochondrial electron transport chain.

The charge/oxygen (q+/O) stoichiometry of mitochondria respiring on succinate was measured under conditions of high membrane potential (delta psi). The technique used was a variation of the steady-state method of Al-Shawi and Brand [(1981) Biochem. J. 200, 539-546]. We show that q+/O was about 2.7 at high values of delta psi (170 mV). As delta psi was lowered from 170 mV to 85 mV with the respiratory inhibitor malonate the q+/O stoichiometry increased to 6.0. A number of artefacts which could have led to an underestimation of the q+/O stoichiometry were eliminated. These included effects of any rapid change in mitochondrial volume, internal pH, activity of the endogenous K+/H+ exchanger or in H+ conductance due to changes in delta psi after the addition of inhibitor. The experiments presented here are the first direct demonstration that the stoichiometry of proton pumping by the mitochondrial respiratory chain changes as delta psi is varied.

Animals↗

PubMed1992

Stoichiometry of interaction between interferon gamma and its receptor.

The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein. The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering. Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein.

Amino Acid Sequence↗

PubMed2000

Stoichiometry of Na+-Ca2+ exchange in inside-out patches excised from guinea-pig ventricular myocytes.

1. The stoichiometry (nx) of cardiac Na+-Ca2+ exchange was examined by measuring the reversal potential of the Na+-Ca2+ exchange current (INa-Ca) in large inside-out patches, 'macro patches', excised from intact guinea-pig ventricular cells. 2. Cytoplasmic application of Na+ (Na+i) or Ca2+ (Ca2+i) induced INa-Ca which showed properties similar to INa-Ca in the giant membrane patch. The outward INa-Ca was depressed by an exchanger inhibitory peptide, XIP. 3. The reversal potential of the XIP-sensitive current indicated that nx was approximately 4 (3.6-4.2) at 9-40 mM Na+i, and nx tended to increase as Na+i was increased. Proteolysis by trypsin did not significantly affect the stoichiometry. Similar results were obtained from the reversal potential of INa-Ca that was induced by application of both Na+i and Ca2+i. 4. At 0.1 microM Ca2+i, nx was approximately 4 (3.7-4. 4) at 6-25 mM Na+i and tended to increase as Na+i was increased. When Ca2+i was changed from 0.1 to 1 and 1000 microM at constant 50 mM Na+i, the value was approximately 4 (3.6-4.4). 5. When the extracellular Na+ (Na+o) and Ca2+ (Ca2+o) concentrations were varied in the presence of 25 or 9 mM Na+i and 1 microM Ca2+i, nx was almost constant ( approximately 4) over the range 0.3-20 mM Ca2+o and 10-145 mM Na+o. 6. These results indicated that the stoichiometry of Na+-Ca2+ exchange is different from generally accepted 3Na+:1Ca2+, and suggested that the stoichiometry is either 4Na+:1Ca2+ or variable depending on Na+i and Ca2+i.

Animals↗

PubMed2001

The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO(3)(-):1 Na(+).

The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3-:Na+ stoichiometry is not larger then 2:However, there are no data available on either the reversal potential or the HCO3-:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current-voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO3-:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3-:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

Animals↗

PubMed2005

Structural evidence for a constant c11 ring stoichiometry in the sodium F-ATP synthase.

The Na+-dependent F-ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria.

Electrophoresis, Polyacrylamide Gel↗

PubMed2003

Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors.

Two functional types of nicotinic acetylcholine receptors (nAChRs) are expressed when human embryonic kidney cells are permanently transfected with equal amounts of human alpha4 and beta2 subunit cDNAs. Most (82%) of these nAChRs exhibit an EC(50) of 74 +/- 6 microM for ACh, a much lower sensitivity than the remaining fraction (EC(50) of 0.7 +/- 0.4 microM) or than expected from expression of equal amounts of alpha4 and beta2 mRNAs in Xenopus laevis oocytes. We have found three conditions that can increase the number of nAChRs with high sensitivity to activation. These are: 1) transient transfection with additional beta2 subunits, 2) overnight incubation in nicotine, or 3) overnight culture at 29 degrees C. Using metabolic labeling with [(35)S]methionine to measure subunit stoichiometry, we found that the majority of nAChRs had a stoichiometry of (alpha4)(3)(beta2)(2). Overnight treatment with nicotine increased the number of nAChRs and increased the proportion of the (alpha4)(2)(beta2)(3) stoichiometry. Alternate alpha4beta2 nAChR stoichiometries with distinct functional properties raise the possibility for an interesting mode of synaptic regulation for nicotinic signaling in the mammalian brain.

Cells, Cultured↗

PubMed1976

Proton movements coupled to lactate and alanine transport in Escherichia coli: isolation of mutants with altered stoichiometry in alanine transport.

The addition of lactate to lightly buffered suspensions of resting cells of Escherichia coli caused an increase in the pH of the extracellular phase as lactate and protons entered the cell together. From the magnitude of the pH change and the non-electrogenic character of lactate uptake, we concluded that the stoichiometry of the process was 1 proton/lactate anion. The addition of alanine caused a slow increase in pH, also apparently due to the transport of the amino acid by a symport mechanism with 1 proton/alanine stoichiometry. When cells were grown in the chemostat with alanine as sole carbon source and as limiting nutrient, this stoichiometry was found to alter to 2 protons/alanine, and then to 4 protons/alanine. These increases stoichiometries were due to the selection of mutants. The consequences of these changes on the potential uptake capacity of the cells are discussed.

Alanine↗

PubMed1984

Sodium-sugar coupling stoichiometry in chick intestinal cells.

Sodium-dependent sugar transport systems involve the function of membrane components that couple the transmembrane flow of Na+ to the concomitant flow of certain sugar molecules. The coupling stoichiometry between Na+ and sugar fluxes via these systems must be measured under conditions in which the membrane potential does not change due to the induction of transport or during the interval of flux measurement. This can be accomplished by utilizing gradients of highly permeant ions (NO-3 and K+ plus valinomycin) to create diffusion potentials of sufficient magnitude that the sugar-induced Na+ flux does not introduce an appreciable change in the imposed potential. Under these conditions, the coupling stoichiometry for chicken intestinal cells proves to be 2 Na+:1 sugar as reported earlier for studies performed in the absence of a membrane potential. When control of the potential is not maintained, a coupling ratio of 1:1 is observed. The stoichiometry does not change as a function of Na+ concentration, which suggests that carrier forms with only one Na+ bound do not contribute to the carrier-mediated Na+ or sugar fluxes. When no potential is present, the stoichiometry is modified by the level of intracellular Na+ and sugar in a manner indicative of a transport mechanism in which Na+ must dissociate from the "loaded" carrier at the inward facing membrane surface before the sugar molecule dissociates.

Adenosine Triphosphate↗

PubMed1987

Stoichiometry of Na+-HCO-3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex.

The major pathway for HCO3- transport across the basolateral membrane of the proximal tubule cell is electrogenic Na+-HCO3- cotransport. In this study, we have determined the stoichiometry of the Na+-HCO3- cotransport system in basolateral membrane vesicles that were isolated from rabbit renal cortex by Percoll gradient centrifugation. When the membrane potential is approximated by the Nernst potential for K+, as in the presence of the K+ ionophore valinomycin, equilibrium thermodynamics predicts that the Na+-HCO3- cotransport system should come to equilibrium and mediate no net flux when (Na)i/(Na)o = [(HCO3)o/(HCO3)i]n[(K)o/(K)i]n-1, where n is the HCO3-:Na+ stoichiometry. Our experimental approach was to impose transmembrane Na+, HCO3-, and K+ gradients of varying magnitude and direction, and then to measure the net flux of Na+ over the subsequent 3-s period. In this way, we could determine the conditions for equilibrium of the transport system and thereby calculate n. The results of these experiments indicate that the value of n is greater than 2.6 and less than 3.5, consistent with a stoichiometry of 3 HCO3-:1 Na+, or a thermodynamically equivalent process. Based on reported intracellular potentials and ion activities, this value for the stoichiometry indicates that the inside-negative membrane potential is sufficient to drive HCO3- exit against the inward concentration gradients of HCO3- and Na+ that are present across the basolateral membrane of the intact proximal tubule cell under physiologic conditions.

Animals↗

PubMed1999

Stoichiometry of the leaching process of fluoride-containing aluminosilicate glass-ionomer glasses.

Dental glass-ionomer cements (GIC) set by an acid-base reaction between a polyalkenoic acid and an ion-leachable glass. The exact relationship between the glass composition and the setting and final properties of GIC is not yet fully elucidated. As part of a systematic study of this relationship, we studied the leaching stoichiometry of glasses used in commercial formulations to correlate the glass composition with its leaching properties. The leaching experiments were performed in acetic acid solutions at pH = 3.4 by means of a pH-stat method. After predetermined time intervals, the suspension was filtered and the filtrate was analyzed for the glass constituents. The usefulness of the pH-stat method for the determination of glass reactivity was corroborated. The deviation of the leaching stoichiometry with respect to the pure glass stoichiometry decreased with increasing relative content of mono- and bivalent glass network dwellers and modifiers. Indications were found that the latter can be leached out independently and preferentially, while the leaching of network dwellers is coupled with the aluminum release. The F content as well as the reactivity of the glass affect the amount of fluoride available for release from a set GIC. It could be concluded that the leaching stoichiometry of GIC glasses can be correlated with their absolute and relative composition.

Acetates↗

PubMed2005

Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein.

GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of alpha, beta, and gamma subunits can be confounded by receptors expressed from alpha and beta subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" alpha1 subunit linked to a beta2 subunit. Cotransfection of the gamma2 subunit with alphabeta-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the gamma subunit with nonlinked alpha1 and beta2 subunits. Similarly, expression of alpha subunits together with alphabeta-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of alpha with beta subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of alphabeta-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of alpha1beta2 and alpha1beta2gamma2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in alpha1beta2gamma2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of alphabetagamma receptors.

Adaptor Proteins, Signal Transducing↗