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F M Harold

Publications and source records attributed to F M Harold.

At least 37 records · Page 2Linked to original sources
PubMed1984

Transcellular ion currents in the water mold Achlya. Amino acid proton symport as a mechanism of current entry.

Achlya, like other tip-growing organisms, generates an endogenous electrical current such that positive charge flows into the hyphal apex and exits from the trunk. The present study is concerned with the mechanism of current generation by hyphae growing in a defined, complete medium. The intensity of the current, measured in the extracellular medium with a vibrating probe, was unaffected by the removal of all the inorganic constituents of the growth medium. However, an increase in the external pH or the deletion of amino acids abolished the current. Removal of methionine alone diminished the current by two thirds. Hyphae also generated a longitudinal pH gradient in the extracellular medium; the region surrounding the tip was more alkaline than the bulk medium, whereas the region around the trunk was relatively acidic. These findings suggest that a flux of protons, dependent upon amino acids in the medium, carries current into the tip and creates the surrounding alkaline zone. The proton current appears to result from the transport of amino acids rather than their metabolism. Conditions that abolished the current also inhibited methionine uptake but had little effect on the respiratory rate. The findings imply a connection between the proton current and chemiosmotic energy transduction. We propose that protons flow into the hyphal tip through amino acid/proton symporters that are preferentially localized there. The proton flux energizes the uptake of amino acids into the growing zone and may also contribute to the polarization of hyphal growth.

Amino Acids↗
PubMed1984

Growing hyphae of Achlya bisexualis generate a longitudinal pH gradient in the surrounding medium.

Growing hyphae of Achlya bisexualis were found to generate a longitudinal pH gradient in the surrounding medium; the medium adjacent to the tip was slightly more alkaline than the bulk phase, while that near distal parts was acidic. The profile of external pH paralleled that of electric current, as measured with a vibrating probe; the apical alkaline zone corresponded to the region of current inflow. In organisms grown in complete medium, both current flow and apical alkalinization were inhibited when amino acid uptake was blocked, either by removing amino acids from the medium or by raising the external pH to 8.5. Achlya could, however, adapt to a medium deficient in organic nutrients; elongating hyphae again generated both the pH profile and the transcellular electric current. It is proposed that both the pH profile and the electric current are manifestations of a transcellular proton current, which arises from the segregation of proton pumps from proton leaks. Symport of protons with amino acids may be one mechanism by which protons enter the hyphal apex.

Adaptation, Physiological↗
PubMed1982

ATP-driven sodium pump in Streptococcus faecalis.

Sodium extrusion by bacteria is generally attributed to secondary antiport of Na+ for H+ energized by the proton circulation. Streptococcus faecalis is an exception, in that sodium expulsion from intact cells requires the generation of ATP but does not depend on the protonmotive force. Unfortunately, studies with everted membrane vesicles failed to reveal the expected sodium pump; instead, the vesicles contained a conventional secondary Na+/H+ antiporter. We report here that everted membrane vesicles prepared in the presence of protease inhibitors retain an ATP-driven sodium transport system. The evidence includes the findings that (i) accumulation of 22Na+ by these vesicles is resistant to reagents that dissipate the protonmotive force but requires ATP and (ii) the vesicles contain a sodium-stimulated ATPase that is distinct from F1F0 ATPase, and whose presence is correlated with sodium transport activity. Sodium movements appear to be electroneutral and are accompanied by movement of H+ in the opposite direction. When membranes are incubated in the absence of protease inhibitors, a secondary Na+/H+ antiport activity emerges, possibly by degradation of the sodium pump. We suggest that S. faecalis expels Na+ by means of an ATP-driven primary transport system that mediates exchange of Na+ for H+. The Na+/H+ antiporter seen in earlier membrane preparation is an artefact of proteolytic degradation.

Adenosine Triphosphatases↗
PubMed1982

Circulation of potassium across the plasma membrane of Blastocladiella emersonii: K+ channel.

A previous paper reported that the water mold Blastocladiella emersonii generates a transcellular electrical current, such that positive charges enter the rhizoid and leave from the thallus (Stump et al., Proc. Natl. Acad. Sci. U.S.A. 77: 6673-6677, 1980). To begin to understand the genesis of this current we investigated ionic relationships in this organism by use of intracellular microelectrodes. In cells suspended in buffered CaCl2, the membrane potential could be accounted for as a K+ diffusion potential; no evidence for an electrogenic pump was obtained. Potassium ions diffuse outward by a pathway that also carries Rb+ and Ba2+, but excludes both smaller and larger ions (Li+, Na+, Cs+, Mg2+, Ca2+, and choline). Chloride and other anions make little contribution to the potential, but the presence of Ca2+ in the external medium is required for successful potential measurements. In growing cells, the internal K+ concentration is generally somewhat higher than would be expected if the K+ distribution were determined entirely by the membrane potential. Under certain conditions, net uptake of K+ against the electrochemical potential gradient was observed. We suggest that K+ is actively accumulated by a primary transport system that may exchange K+ for H+, and that K+ leaks passively outward through the K+ channel. The K+ circulation across the membrane amounts to about 2% of the K+ pool per min, or 4.5 microA/cm2 of surface area. We propose that this K+ circulation is one arm of the transcellular current, carrying positive charge out of the thallus.

Anions↗
PubMed1982

Selective transport of nutrients via the rhizoids of the water mold Blastocladiella emersonii.

Previous work in this laboratory demonstrated that the rhizoids of Blastocladiella emersonii grow chemotropically toward a source of Pi and thus provided preliminary evidence that, in addition to serving as a holdfast, the rhizoids absorb nutrients. To further examine the role of the rhizoids in nutrient uptake, we devised a technique to introduce a barrier between the rhizoids and the thallus to that these cell compartments could be studied independently. Cells were grown on polycarbonate membrane filters in such a way that all of the thalli were on one side of the filter and essentially all of the rhizoids were on the opposite side. Nutrient uptake into the rhizoids and the thallus was measured by floating the filters bearing cells on radioactive medium so that only one side of the filter contacted the label. Mineral oil was used to block the diffusion of the label through the unfilled pores in the filter. This technique permitted us to establish clearly that the rhizoids absorb all seven of the nutrients tested. In addition, we found that some nutrients, specifically Pi and amino acids, appeared to be preferentially taken up via the rhizoids, whereas K+, Rb+, and Ca2+ entered the thallus and rhizoids equally. Cells grown in the presence of the microtubule synthesis inhibitors nocodazole and carbendazim elaborated only a stunted rhizoid system, so we examined their ability to accumulate the two classes of compounds. As expected, these cells were severely inhibited in Pi and amino acid uptake but retained normal uptake of K+, Rb+, and Ca2+.

Biological Transport↗
PubMed1980

ATP-linked sodium transport in Streptococcus faecalis. I. The sodium circulation.

Streptococcus faecalis, like other bacteria, expels Na+ and accumulates K+. Sodium movements in several bacterial species have been attributed to secondary antiport of Na+ for H+, energized by the proton-motive force. We find a more complex pattern: a circulation of Na+ across the plasma membrane. One limb is the diffusion of Na+, into the cells or out, by a low affinity pathway in response to gradients of concentration and of electrical potential; Na+ movements are enhanced in metabolizing cells. The other limb is the vectorial extrusion of Na+ by a transport system that requires "ATP" (either ATP itself or a related metabolite), even when Na+ is moving downhill. Cells glycolyzing at alkaline pH in buffer containing excess K+ can expel Na+ against a concentration gradient of 100-fold, even in the presence of reagents that block or short circuit the proton circulation (pH gradient and membrane potential both zero). Evidently, under these conditions "ATP" can serve as the energy donor for a primary sodium pump. However, at acid pH, or in presence of low levels of K+, sodium extrusion requires both "ATP" and the proton-motive force. A mutant is described that retains the leak pathway but lacks the "ATP"-linked transport system.

Adenosine Triphosphate↗
PubMed1980

Energy coupling to potassium transport in Streptococcus faecalis. Interplay of ATP and the protonmotive force.

We have studied the mechanism by which metabolic energy is coupled to potassium accumulation by the fermentative bacterium, Streptococcus faecalis. In starving cells, K+ movements into the cells or out are very slow; even 42K+/K+ exchange requires concurrent metabolism of glucose or arginine. Metabolizing cells accumulate K+, establishing a concentration gradient of some 50,000. Accumulation is prevented by reagents that block or short circuit the proton circulation, but 42K+/K+ exchange persists. In glycolyzing cells whose proton pump has been blocked with N,N'-dicyclohexylcarbodiimide, net uptake of K+ can be induced by imposing an artificial membrane potential, interior negative. Net K+ efflux is also controlled by the interplay of ATP and the proton circulation. Addition of proton conductors to glycolyzing cells induces K+ efflux, but has no effect on starving cells; the rate of K+ efflux appears to be a function of the cells' ATP content. We conclude that K+ accumulation requires the cells to generate both a protonmotive force and ATP. K+ uptake is electrogenic and attains a concentration gradient far too steep to be in equilibrium with the membrane potential. We consider two alternative models for K+ transport: a primary ATP-driven pump regulated by the proton circulation or a secondary porter activated by ATP that mediates symport of K+ with H+.

Adenosine Triphosphate↗
PubMed1980

Endogenous electrical currents in the water mold Blastocladiella emersonii during growth and sporulation.

We have explored the pattern of electrical currents generated by single cells of the water mold Blastocladiella emersonii at several stages of its life cycle. Extracellular currents were measured with a vibrating probe constructed after the design of Jaffe and Nuccitelli [Jaffe, L. F. & Nuccitelli, R. (1974) J. Cell Biol. 63, 614-628]. In growing cells positive current, of the order of 1 microA/cm2, enters the rhizoid and leaves from the thallus; circumstantial evidence suggests that protons carry much of the current. Sporulation is associated with reversal of the current pattern, such that positive current enters the thallus and leaves from the rhizoidal region; the ions that carry the current have not been identified. These current patterns appear to play a role in the spatial localization of fungal growth and development.

Blastocladiella↗
PubMed1980

Ionic control of germination of Blastocladiella emersonii zoospores.

Encystment and germination of Blastocladiella emersonii zoospores involve a rapid and radical transformation of the motile but nongrowing spore into a sessile, growing germling. Certain inorganic ions, notably 50 mM KCl, are efficient inducers of germination. By use of the carbocyanine dye DiO-C6-(3), we found that KCl depolarizes the plasma membrane of zoospores and noted good correlation between depolarization and subsequent germination. Zoospores avidly accumulated K+ ions from the medium, attaining an internal concentration of over 50 mM and a concentration gradient of 2,500. Sodium ions, by contrast, were expelled. Internal K+ was required for normal germination but its function is not known. Zoospores also took up considerable amounts of calcium; most of this was associated with the external surface and appeared to be necessary for maintenance of zoospore integrity. KCl (50 mM) and other salts displaced surface calcium but this was not in itself sufficient to induce germination. The calcium ionophore A23187, in the presence of external calcium, was an effective inducer of germination, suggesting a possible role for cytosolic calcium in triggering the transformation. We propose that the first step in the induction of germination by salts is depolarization of the plasma membrane; subsequent events require the intervention of cytoplasmic signals.

Blastocladiella↗
PubMed1980

Oriented growth of Blastocladiella emersonii in gradients of ionophores and inhibitors.

To investigate whether ion currents help to localize growth and development of Blastocladiella emersonii, we grew the organisms in gradients of various ionophores and inhibitors. Gradients were generated by placing into the culture fine glass fibers coated with insoluble inhibitors; in some cases, inhibitors were adsorbed onto beads of ion-exchange resin. Organisms growing in many of these gradients exhibited a striking tendency for the thalli to grow toward the fiber. This proved to be misleading; the cells grew not toward the source of the ionophore but into the unoccupied zone of inhibition adjacent to the fiber. Fibers coated with gramicidin-D induced marked effects on the growth of the rhizoids, which were greatly enlarged and grew toward and onto the fiber. None of the other inhibitors produced such effects, except for beads coated with the proton conductors tetrachlorosalicylanilide and compound 1799. The results suggest that orientation of rhizoid growth results from enhancement of proton flux across the plasma membrane. Growth of the rhizoids was also strongly oriented by gradients of inorganic phosphate and an amino acid mixture; gradients of glucose, K+, Ca2+, and glutamate were ineffective. We propose that a major physiological function of the rhizoid is to transport nutrients to the thallus. Finally, we examined the effects of a series of benzimidazole antitubulins as well as the cytochalasins. These did not orient growth but grossly perturbed the pattern of cellular organization, producing small spherical cells with multiple stunted rhizoids. The findings are interpreted in terms of the interaction of an endogenous transcellular proton current with elements of the cytoskeleton in the determination of form.

Amino Acids↗
PubMed1979

Energy coupling to the transport of inorganic phosphate in Escherichia coli K12.

The nature of the energy source for phosphate transport was studied in strains of Escherichia coli in which either one of the two major systems (PIT, PST) for phosphate transport was present. In the PIT system, phosphate transport is coupled to the proton-motive force. The energy source for the PST system appears to be phosphate-bond energy, as has been found in other systems involving binding proteins. High concentration gradients of phosphate (between 100 and 500) are established by both systems.

Aerobiosis↗
PubMed1977

Circulation of H+ and K+ across the plasma membrane is not obligatory for bacterial growth.

Streptococcus faecalis grows normally in the presence of gramicidin and other ionophores under conditions such that there is no gradient of pH or of electrical potential across the plasma membrane and that currents of H+, K+, and Na+ are short-circuited. Growth requires a rich medium, a slightly alkaline pH, and a high concentration of external K+. The proton circulation maintains the cytoplasmic pH and pools of ions and other metabolites but is not obligatory for biosynthetic functions including DNA replication, cell division, or assembly of the structural framework of the cell.

Adenosine Triphosphate↗
PubMed1977

A protonmotive force drives bacterial flagella.

Streptococcus strain V4051 is motile in the presence of glucose. The cells move steadily along smooth paths (run), jump about briefly with little net displacement (twiddle), and then run in new directions. They stop swimming when deprived of glucose. These cells become motile when an electrical potential or a pH gradient is imposed across the membrane. Starved cells suspended in a potassium-free medium respond to the addition of valinomycin by a brief period of vigorous twiddling. They also twiddle, although less vigorously, when the external pH is lowered. Valinomycin-induced twiddling occurs in the absence of external alkali or alkaline earth cations and without significant net synthesis of ATP. When a chemoattractant is added to cells swimming in the presence of glucose, twiddles are transiently suppressed, and the cells run for a time. Similarly, when starved cells are suspended in a potassium-free medium containing both valinomycin and an attractant, many cells initially run rather than twiddle. We conclude that the flagella are driven by a protonmotive force.

Adenosine Triphosphate↗
PubMed1975

Accumulation of lipid-soluble ions and of rubidium as indicators of the electrical potential in membrane vesicles of Escherichia coli.

We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration. Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin. By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces. Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain. Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles. Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation. Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles. Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups. This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+. Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+. Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport. Proton-conducting ionophores and several inhibitors of respiration block Rb+ uptake and dissipated a preformed gradient. Preincubation of the vesicles with oxamate or p-chloromercuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ complete for uptake when present simultaneously. We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration.

Antimetabolites↗