Pseudomonas aeruginosa cytochrome oxidase. Product inhibition by low thermodynamic driving force

The steady-state kinetics of Pseudomonas aeruginosa cytochrome oxidase were studied. Reduced cytochrome c551 and azurin from the same bacteria were used as the electron-donating substrates, while dioxygen served as the electron acceptor. Oxidized cytochrome c551 and azurin exhibited product inhibition of the reaction. However, apo-azurin and azurin derivatives in which the copper was substituted by the redox-inert ions Ni2+, Co2+, Cd2+ and Zn2+, did not show any effect on the kinetics. These observations implied that complex formation between the substrates or the products and the enzyme is not a rate-limiting step and is not the cause for product inhibition. The integrated rate law for a reaction scheme in which we assumed that complex formation was not rate limiting was fitted to the complete reaction traces. The results suggested that it is the low thermodynamic driving force, expressed in the small differences in redox potential between the substrates and heme c of the enzyme, which cause the observed product inhibition.     

Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy

The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission intensities and anisotropies of eosin and erythrosin immobilized in poly(methyl methacrylate). The fluorescence data are compared with those from phosphorescence emission measurements and with theoretical predictions. Triplet-state lifetimes were obtained in 5 mM phosphate buffer, pH 7.0, of concanavalin A labeled with eosin, tetramethylrhodamine, and fluorescein and of alpha 2-macroglobulin labeled with the first two probes. In the case of labeled concanavalin A, iodide quenching measurements gave bimolecular rate constants of approximately 10(9) M-1 s-1. The usefulness of FRS for studying protein-cell interactions is exemplified with eosin-labeled concanavalin A bound to living A-431 human epidermoid carcinoma cells. Finally, the advantages and disadvantages of the technique are compared to those of the alternative phosphorescence emission method.     

Mechanisms of fusicoccin action: A dominant role for secondary transport in a higher-plant cell

Fusicoccin (FC) is commonly thought to promote electrogenic H⁺ extrusion through its action on the H⁺-ATPase of the plant plasma membrane. Nonetheless, essential support from rigorous electrophysiological analysis has remained largely absent. The present investigation surveys the effects of FC on the charge transport properties at the membrane of a higher-plant cell — stomatal guard cells of Vicia faba L. — for which the electrical geometry is defined, and from which the voltage-dependent kinetic characteristic for the pump has been identified. Current-voltage (I-V) relations of the guard cells were determined before and during treatments with FC, and during brief exposures to NaCN plus salicylhydroxamic acid. Responses of the pump and of the ensemble of secondary transport processes were identified in the whole-membrane conductance-voltage relations and in the difference-current-voltage (dI-V) characteristic for the pump. In 0.1 mM K⁺, exposure to 10 M FC shifted guard-cell potentials negative by 29–61 mV. Current-and conductance-voltage profiles indicated limited changes in the pump I-V characteristic, an observation which was confirmed through explicit kinetic analysis of pump dI-V relations. However, the voltage response was accompanied by a 1.5-to 2.6-fold fall in membrane conductance. These results challenge conventional views of fusicoccin action by ascribing the electrical responses to reduced current passage through secondary transport pathways as well as to enhanced electrogenic ion pumping.Abbreviations and symbols Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SHAM salicylhydroxamic acid - FC fusicoccin - V _m free-running membrane potential - G _m membrane slope conductance at V _m - (d)I-V (difference) current-voltage (relation) - G-V slope conductance-voltage (relation)     

A potassium-proton symport in Neurospora crassa

Combined ion flux and electrophysiological measurements have been used to characterized active transport of potassium by cells of Neurospora crassa that have been moderately starved of K+ and then maintained in the presence of millimolar free calcium ions. These conditions elicit a high-affinity (K1/2 = 1-10 microM) potassium uptake system that is strongly depolarizing. Current-voltage measurements have demonstrated a K+-associated inward current exceeding (at saturation) half the total current normally driven outward through the plasma membrane proton pump. Potassium activity ratios and fluxes have been compared quantitatively with electrophysiological parameters, by using small (approximately 15 micron diam) spherical cells of Neurospora grown in ethylene glycol. All data are consistent with a transport mechanism that carries K ions inward by cotransport with H ions, which move down the electrochemical gradient created by the primary proton pump. The stoichiometry of entry is 1 K ion with 1 H ion; overall charge balance is maintained by pumped extrusion of two protons, to yield a net flux stoichiometry of 1 K+ exchanging for 1 H+. The mechanism is competent to sustain the largest stable K+ gradients that have been measured in Neurospora, with no direct contribution from phosphate hydrolysis or redox processes. Such a potassium-proton symport mechanism could account for many observations reported on K+ movement in other fungi, in algae, and in higher plants.     

One heavy chain variable region gene segment subfamily in the BALB/c mouse contains 500-1000 or more members

Nucleic acid hybridization studies suggest that 500-1000 or more heavy chain variable (VH) gene segments related to one VH gene segment (J558) are present in the genome of the BALB/c mouse. The implications of this result regarding the overall size, sequence organization, and evolution of the mouse family of VH gene segments are discussed.     

Voltage dependence of theChara proton pump revealed by current-voltage measurement during rapid metabolic blockade with cyanide

Summary It is generally agreed that solute transport across theChara plasma membrane is energized by a proton electrochemical gradient maintained by an H⁺-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H⁺-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the trueI-V profile for the pump when treatments alter the thermodynamic pressure on transport.We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cellI-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl₃, so that steady-state currents could be recorded under voltage clamp between –400 and +100 mV. Exposures to 1mm NaCN (CN) and 0.4mm salicylhydroxamic acid (SHAM) depolarized (positive-going)Chara membrane potentials by 44–112 mV with a mean half time of 5.4±0.8 sec (n=13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3±0.9 sec ([ATP] _t=0, 0.74±0.3mm; [ATP] _t=x , 0.23±0.02mm). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses ofdI-V curves (±CN+SHAM) as well as of families ofI-V curves taken at times during CN+SHAM exposures indicated a stoichiometry for the pump of one charge (H⁺) transported per ATP hydrolyzed and an equilibrium potential near –420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60–75% of the total membrane conductance nearV _m. Complementary results were obtained also in fitting previously publishedI-V data gathered over the external pH range 4.5–7.5 Kinetic features deduced for the pump were dominated by a slow step preceding H⁺ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium. These characteristics predict, in marked contrast to the situation forNeurospora, that cytoplasmic acid loads inChara should shift the pumpI-V curve negative-going along the voltage axis with little change in maximum current output at positive voltages.     

Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid

Evidence of a role for abscisic acid (ABA) in signalling conditions of water stress and promoting stomatal closure is convincing, but past studies have left few clues as to its molecular mechanism(s) of action; arguments centred on changes in H⁺-pump activity and membrane potential, especially, remain ambiguous without the fundamental support of a rigorous electrophysiological analysis. The present study explores the response to ABA of K⁺ channels at the membrane of intact guard cells ofVicia faba L. Membrane potentials were recorded before and during exposures to ABA, and whole-cell currents were measured at intervals throughout to quantitate the steady-state and time-dependent characteristics of the K⁺ channels. On adding 10 M ABA in the presence of 0.1, 3 or 10 mM extracellular K⁺, the free-running membrane potential (V _m) shifted negative-going (–)4–7 mV in the first 5 min of exposure, with no consistent effect thereafter. Voltage-clamp measurements, however, revealed that the K⁺-channel current rose to between 1.84- and 3.41-fold of the controls in the steady-state with a mean halftime of 1.1 ± 0.1 min. Comparable changes in current return via the leak were also evident and accounted for the minimal response inV _m. Calculated atV _m, the K⁺ currents translated to an average 2.65-fold rise in K⁺ efflux with ABA. Abscisic acid was not observed to alter either K⁺-current activation or deactivation.These results are consistent with an ABA-evoked mobilization of K⁺ channels or channel conductance, rather than a direct effect of the phytohormone on K⁺-channel gating. The data discount notions that large swings in membrane voltage are a prerequisite to controlling guard-cell K⁺ flux. Instead, thev highlight a rise in membranecapacity for K⁺ flux, dependent on concerted modulations of K⁺-channel and leak currents, and sufficiently rapid to account generally for the onset of K⁺ loss from guard cells and stomatal closure in ABA.