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C L Slayman

Publications and source records attributed to C L Slayman.

At least 55 records · Page 3Linked to original sources
PubMed1981

Interpretation of current-voltage relationships for "active" ion transport systems: I. Steady-state reaction-kinetic analysis of class-I mechanisms.

This paper develops a simple reaction-kinetic model to describe electrogenic pumping and co- (or counter-) transport of ions. It uses the standard steady-state approach for cyclic enzyme- or carrier-mediated transport, but does not assume rate-limitation by any particular reaction step. Voltage-dependence is introduced, after the suggestion of Läuger and Stark (Biochim. Biophys. Acta 211:458-466, 1970), via a symmetric Eyring barrier, in which the charge-transit reaction constants are written as k12 = ko12 exp(zF delta psi/2RT) and k21 = ko21 exp(-zF delta psi/2RT). For interpretation of current-voltage relationships, all voltage-independent reaction steps are lumped together, so the model in its simplest form can be described as a pseudo-2-state model. It is characterized by the two voltage-dependent reaction constants, two lumped voltage-independent reaction constants (k12, k21), and two reserve factors (ri, ro) which formally take account of carrier states that are indistinguishable in the current-voltage (I-V) analysis. The model generates a wide range of I-V relationships, depending on the relative magnitudes of the four reaction constants, sufficient to describe essentially all I-V datas now available on "active" ion-transport systems. Algebraic and numerical analysis of the reserve factors, by means of expanded pseudo-3-, 4-, and 5-state models, shows them to be bounded and not large for most combinations of reaction constants in the lumped pathway. The most important exception to this rule occurs when carrier decharging immediately follows charge transit of the membrane and is very fast relative to other constituent voltage-independent reactions. Such a circumstance generates kinetic equivalence of chemical and electrical gradients, thus providing a consistent definition of ion-motive forces (e.g., proton-motive force, PMF). With appropriate restrictions, it also yields both linear and log-linear relationships between net transport velocity and either membrane potential or PMF. The model thus accommodates many known properties of proton-transport systems, particularly as observed in "chemiosmotic" or energy-coupling membranes.

Animals↗
PubMed1981

Role of the plasma membrane proton pump in pH regulation in non-animal cells.

Possible methods by which eukaryotic cells can regulate intracellular pH (pHi) in response to experimental acid loading were investigated by using as a model cell the fungus Neurospora. Attention was focused on the role of membrane transport in such regulation, starting from the fact that this organism possesses a powerful electrogenic proton extrusion pump. Intracellular acidification was forced by introducing butyric acid into the recording medium, and subsequent changes in pHi and membrane potential were determined with intracellular microelectrodes. In separate experiments, membrane current-voltage curves were obtained and resolved--by an explicit kinetic model--into distinct pump and leak components. Decreased pHi causes increased outward pumping of H+ ions, in a manner quantitatively consistent with their role as a substrate for the proton pump. This increased pumping is often manifest as a transient hyperpolarization at the onset of cytoplasmic acidification. With a considerably slower time course, decreased pHi also produces a large increase in membrane leak conductance, which brings about net membrane depolarization and further stimulates the pump (by virtue of the reduced back electromotive force). Although the identity of the ion responsible for increased leak conductance is not yet known, the evident modulation of conductance seemingly plays an important role in stabilizing the intracellular pH: Stimulation of the pump alone would have little net effect on pHi because it would result simply in enhanced backflux of H+ (to which the membrane is most permeable in normal circumstances). An increased leak to nonprotons, however, would allow the pump to accomplish net H+ ejection.

Acid-Base Equilibrium↗
PubMed1980

Quantitative measurements of membrane potential in Escherichia coli.

By use of giant cells of Escherichia coli induced by growth in the presence of 6-amidinopenicillanic acid, membrane potentials have been measured by two completely independent techniques: directly with intracellular microelectrodes and indirectly from the steady-state distribution of [3H]tetraphenylphosphonium. Under a variety of conditions, the two methods yield values that agree very closely. Thus, with both techniques, the membrane potential approximates -85 mV (interior negative) at pH 5.0 and -142 mV at pH 8.0, with an average slope of -22 mV/pH unit over the range pH 5.0-7.0. A parallel study of membrane vesicles prepared from giant cells was undertaken using tetraphenylphosphonium distribution alone as a measure of membrane potential. The vesicles were found to exhibit a much smaller slope of membrane potential vs. extracellular pH (about -6 mV/pH unit) than intact giant cells. The results indicate that distribution studies with these lipophilic cations provide an excellent measure of membrane potential and are discussed in relation to calculations of H+/substrate stoichiometry for protonsymport systems in E. coli.

Amdinocillin↗
PubMed1980

Metabolic modulation of stoichiometry in a proton pump.

The current-voltage characteristics of the ATP-dependent proton pump in the plasma membrane of Neurospora have been explored under varied metabolic conditions imposed by mutation and by differential respiratory inhibition. The reversal potential, or presumed equilibrium potential, for the pump was observed at about -400 mV under energy-replete conditions, and at about -200 mV during a stable metabolic downshift of 55 percent. Steady-state levels of adenine nucleotides and inorganic phosphate, however, were not affected by this partial energy restriction, so that under both normal and restricted conditions the apparent free energy of ATP hydrolysis remained near -500 mV. The results suggest that a normal pump stoichiometry of 1 H+ extruded/1 ATP split is modified to 2 H+/1 ATP, by chronic energy restriction.

Adenine Nucleotides↗
PubMed1978

Current-voltage relationships for the plasma membrane and its principal electrogenic pump in Neurospora crassa: I. Steady-state conditions.

The nonlinear membrane current-voltage relationship (I-V curve) for intact hyphae of Neurospora crassa has been determined by means of a 3-electrode voltage-clamp technique, plus "quasi-linear" cable theory. Under normal conditions of growth and respiration, the membrane I-V curve is best described as a parabolic segment convex in the direction of depolarizing current. At the average resting potential of - 174 mV, the membrane conductance is approximately 190 micronhos/cm2; conductance increase to approximately 240 micronhos/cm2 at -300 mV, and decreases to approximately 130 micronhos/cm2 at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range. Inhibition of the primary electrogenic pump in Neurospora by ATP withdrawal (with 1 mM KCN) depolarizes the membrane to the range of -40 to -70 mV and reduces the slope of the I-V curve by a fixed scaling factor of approximately 0.8. For wild-type Neurospora, compared under control conditions and during steady-state inhibition by cyanide, the I-V difference curve--presumed to define the current-voltage curve for the electrogenic pump--is a saturation function with maximal current of approximately 20 muA/cm2, a half saturation potential near -300 mV, and a projected reversal potential of ca. -400 mV. This value is close to the maximal free energy available to the pump from ATP hydrolysis, so that pump stoichiometry must be close to 1 H+ extruded:1 ATP split. The time-courses of change in membrane potential and resistance with cyanide are compatible with the steady-state I-V curves, under the assumption the cyanide has no major effects other than ATP withdrawal. Other inhibitors, uncouplers, and lowered temperature all have more complicated effects. The detailed temporal analysis of voltage-clamp data showed three time-constants in the clamping currents: one of 10 msec, for charging the membrane capacitance (0.9 muF/cm/2); a second of 50-75 msec; and a third of 20-30 sec, perhaps representing changes of intracellular composition.

Biological Transport, Active↗
PubMed1978

Production of giant cells of Escherichia coli.

Giant cells, with volumes up to 500-fold those of normal cells, have been produced by both genetic and pharmacological means in Escherichia coli K-12. In the genetic approach, an envB or mon mutation (conferring rounded or irregular morphology) was combined with a lon mutation (block of septation after irradiation). UV irradiation and subsequent incubation for 2 to 5 h in a rich medium supplemented with 1% sodium chloride led t; production of polymorphic giant cells. In the pharmacological approach, incubation of several different strains of E. coli K-12 with the drug 6-amidinopenicillanic acid (FL1060) in the same rich medium gave rise to a homogeneous population of smoothly rounded giant cells.

Cell Division↗
PubMed1976

"Action potentials" in Neurospora crassa, a mycelial fungus.

Occasional spontaneous "action potentials" are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5-20 mV depolarized from the normal resting potential (near-180 mV). The "action potentials" are long-lasting, 1-2 min and at the peak reach a membrane potential near-40 mV. A 2-to 8-fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H+ or Cl- and (b) periodic decreases in activity of the major electrogenic pump (H+) or the Neurospora membrane, coupled with a nonlinear (inverse signoid) current-boltage relationship. Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.

Action Potentials↗
PubMed1975

Oscillations of an electrogenic pump in the plasma membrane of Neurospora.

The presence of the poky mutation in Neurospora crassa produces mitochondria which are defective in cytochromes b and aa3 but which compensate by means of an alternate, cyanide-insensitive oxidase. As previously reported (Slayman, Rees, Orchard & Slayman, J. Biol. Chem., 250:396, 1975) cyanide blockade of the poky strain carrying the partial suppressor f results in a metabolic downshift of only 56%, compared with a downshift of 98% in wild-type Neurospora; the downshift is accompanied by exponential decay of ATP in the wild type, but by an undershoot and monotonic recovery of ATP in poky f. Whereas the membrane potential declines with ATP in wild-type Neurospora, it oscillates near the resting level (ca. -- 185 mV) in poky f. Oscillations begin with a depolarizing swing of 30--100 mV, followed by slight hyperpolarization, then by 2--4 damped cycles having a frequency near 1/min. Similar oscillations arise with antimycin, salicyl hydroxamic acid, and several uncoupling agents, and depend on partial maintenance of respiration through either the defective cytochrome chain or the alternate oxidase. Small oscillations (maximally +/- 30% of the control value) in membrane conductance also occur, roughly in phase with the oscillations of membrane potential. The amplitude of these, in comparison with the nonlinearity of the normal current-voltage relationship for the membrane, strongly suggests that they arise as a secondary consequence of the voltage changes. Therefore, since it has previously been argued (Slayman, Long & Lu, J. Membrane Biol. 14:305, 1973) that most of the resting membrane potential in the organism arises from active extrusion of H+ ions, the simolest interpretation of the cyanide-induced voltage oscillations is that current through the H+ pump is modulated cyclically. The ultimate mechanism for this modulation is unresolved, but could plausible involve a metabolic feedback system, oscillations of intracellular pH, or both. In many respects the observed voltage oscillations resemble the well-known oscillations of mitochondrial H+ flux which are produced by sudden metabolic shifts.

Adenosine Triphosphate↗
PubMed1975

Generation of adenosine triphosphate in cytochrome-deficient mutants of Neurospora.

The fungus Neurospora crassa is known to possess a branched respiratory system consisting of the standard cytochrome chain and a cyanide-insensitive alternate oxidase. In the present experiments, the physiological function of the alternate oxidase has been analyzed by taking advantage of a number of cytochrome-deficient mutants, particularly poky f. Respiration, cellular ATP levels, and growth have been examined under the influence of three classes of inhibitors: inhibitors of the cytochrome chain (antimycin, cyanide), an inhibitor of the laternate oxidase (salicyl hydroxamic acid), and an uncoupling agent (carbonyl cyanide m-chlorophenylhydrazone). The results indicate that the over-all efficiency of the alternate oxidase in producing ATP and supporting growth is much less than that of the cytochrome chain. Depending upon the amount of oxidative phosphorylation at Sites II and III in the cytochrome chain, which varies from strain to strain, the efficiency of the alternate oxidase relative to that of the cytochrome chain ranges from 13% in wild type Neurospora to 18 to 21% in poky f, 35% in mi-3, and 57% in cyt-2. A comparison of the short term effects of cyanide and carbonyl cyanide m-chlorophenylhydrazone on cellular ATP in poky f suggests that, during respiration through the alternate oxidase, ATP can be produced both by substrate-level phosphorylation (accompanying glycolysis and the oxidation of alpha-ketoglutarate) and by oxidative phosphorylation at Site I. When cells are grown on sucrose, as much as 22% of ATP synthesis in the presence of cyanide occurs at Site I. When cells are grown on acetate to diminish the rate of glycolysis, the contribution of Site I becomes proportionately larger. Both the growth experiments and the short term inhibitor experiments reveal that ATP levels in Neurospora are kept high be a feedback process which depresses ATP breakdown (and growth) very quckly after ATP synthesis is inhibited. Thus, poky f grows more slowly that wild type Neurospora and is inhibited still further when either the cytochrome chain or the alternate oxidase is blocked. Under all of these conditions, however, cellular ATP in poky f is maintained at a high level (about 3 mmol per kg of cell water, slightly above the values measured in the wild type strain). Continue.

Acetates↗
PubMed1974

Depolarization of the plasma membrane of Neurospora during active transport of glucose: evidence for a proton-dependent cotransport system.

Intracellular microelectrodes were used to measure the effects of glucose transport on membrane voltage and membrane resistance in Neurospora crassa. Sudden activation of glucose uptake, via the high-affinity, derepressible system II, results in a very large depolarization of the plasma membrane. At saturating concentrations of glucose, the depolarization averages 120 mV; it is diphasic in time, with an initial shift at rates of 100-200 mV/sec followed by a slow, spontaneous, partial repolarization. Changes in intracellular ATP concentration are small and could account for only 10 mV of the initial depolarization, while the rest appears to depend upon the transport process itself. A plot of peak depolarization against the extracellular glucose concentration gives a saturation curve which is half-maximal at 42 muM, in good agreement with the K(1/2) reported for glucose transport via system II. The nonmetabolized analogue 3-O-methyl-D-glucose also causes depolarization, and in addition leads to a pulsed alkalinization of the medium occurring at approximately the same rate as 3-O-methyl-D-glucose uptake. The membrane resistance falls only slightly during glucose depolarization, a fact which requires the transport system itself to have a high internal resistance, or the membrane current-voltage relationship in glucose-starved cells to be quite nonlinear. All of the data support Mitchell's notion that sugar and hydrogen ions are contransported under the influence of the membrane potential, and lead to values for H(+):glucose stoichiometry of 0.8 to 1.4.

Adenosine Triphosphate↗
PubMed1973

Adenine nucleotide levels in Neurospora, as influenced by conditions of growth and by metabolic inhibitors.

By a combination of luciferase and fluorescence methods adenine nucleotide pools in Neurospora crassa have been examined under various conditions of growth and metabolic inhibition. During sustained exponential growth (25 C, shaking liquid cultures), the intracellular adenosine 5'-triphosphate (ATP) concentration, [ATP](i), rises slowly from the conidial level near 1 mM (1 mmol/kg of cell water) to a maximum of 2.0 to 2.5 mM at 14 h, after which it slowly declines. The adenosine 5'-diphosphate and adenosine 5'-monophosphate (AMP) curves show two peaks, at 8 and 20 h, with a minimum at 16 h. The "energy charge" function varies around a mean of 0.72 throughout the period of exponential growth. Transferral of growing cells to buffer lacking a nitrogen source stabilizes the [ATP](i) near 2.5 mM, apparently independent of the cell age, and most studies of metabolic inhibitors were carried out on cells grown 14 to 16 h and then shifted to N-free buffer. Under these conditions sudden respiratory blockade (cyanide) produces exponential decay of ATP with a time constant of about 5.7 s (half-time of 3.9 s), and at a rate which implies a minimal ATP turnover of 0.44 mM/min. This figure is about one-third the rate (1.17 mM/min) which would be calculated from steady-state respiration, a discrepancy which may partly be accounted for by transphosphorylation from appreciable amounts of non-adenine nucleoside di- and triphosphates present in Neurospora. For all three adenine nucleotides, the transients associated with sudden respiratory blockade include overshoots or undershoots of several minutes duration, which are consistent with feedback regulation of glycolysis by the AMP/ATP ratio.

Adenosine Diphosphate↗
PubMed1970

Potassium transport in Neurospora. Evidence for a multisite carrier at high pH.

At low extracellular pH (4-6), net uptake of potassium by Neurospora is a simple exponential process which obeys Michaelis kinetics as a function of [K](o). At high pH, however, potassium uptake becomes considerably more complex, and can be resolved into two distinct exponential components. The fast component (time constant = 1.2 min) is matched quantitatively by a rapid loss of sodium; it is attributed to ion exchange within the cell wall, since it is comparatively insensitive to low temperature and metabolic inhibitors. By contrast, the slower component (time constant = 10.9 min) is inhibited markedly at 0 degrees C and by CN and deoxycorticosterone, and is thought to represent carrier-mediated transport of potassium across the cell membrane. This transport process exhibits sigmoid kinetics as a function of [K](o); the data can be fitted satisfactorily by two different two-site models (one involving a carrier site and a modifier site, the other an allosteric model). Either of these models could also accommodate the simple Michaelis kinetics at low pH.

Biological Transport, Active↗