Amino acid uptake by Saccharomyces cerevisiae plasma membrane vesicles

A procedure is described which allows for the efficient separation of Saccharomyces cerevisiae plasma membranes from other cellular membranes by discontinuous sucrose density gradient centrifugation. After vesiculization in an osmotic stabilization buffer the plasma membrane vesicles retain the ability to transport amino acids. Amino acid uptake was affected by the proton gradient dissipator $\text{m-}\text{chlorocarbonylcyanide}$ phenylhydrazone and was dependent, in some cases, on the presence of sodium ion.     

The influence of uncouplers on proton-sugar symport in Saccharomyces fragilis

Uncouplers of oxidative phosphorylation inhibit proton-sugar symport in Saccharomyces fragilis. However, they do not induce efflux of accumulated sugar. It is shown that the effect cannot be explained by uncoupler-induced alterations in the transmembrane potential or transmembrane pH difference. It is also indicated that a decrease in intracellular pH is not involved in inhibition of sugar transport. It is argued that inhibition of transport by uncouplers is most likely caused by a direct interaction with the translocator.     

Relation of aerobiosis and ionic strength to the uptake of dihydrostreptomycin in Escherichia coli

Aminoglycoside antibiotics exhibit a markedly reduced antibacterial activity under anaerobic conditions. Anaerobiosis or inhibitors of electron transport produced an extensive decrease in the uptake of dihydrostreptomycin in Escherichia coli K-12. Uptake of proline or putrescine were only slightly impaired under anaerobic conditions in the presence of glucose. Both the susceptibility to and the uptake of dihydrostreptomycin under anaerobic conditions were partially restored by addition of the alternative electron acceptor, nitrate. This stimulation required functional nitrate reductase activity. Abolition of uptake by 2,4-dinitrophenol under both aerobic and anaerobic conditions indicates that streptomycin uptake requires electron transport as well as a sufficient membrane potential. In addition, the initial rate of dihydrostreptomycin uptake was competitively and reversibly inhibited by added salts. The inhibition was relatively nonspecific with respect to the identity of salt added, being approximately dependent on the ionic strength. Although dihydrostreptomycin and polyamines mutually inhibited each other's uptake, several conditions (polyamine limitation, streptomycin uptake-deficient mutants) were found in which uptake of these two substrates was oppositely affected. Aminoglycosides thus do not appear to enter on one of the usual cellular transport systems, but perhaps utilize a component of the electron transport system.     

Errata

Mutants of Escherichia coli K-12 which are defective in components of transport systems for uracil and uridine were isolated and utilized to characterized the transport mechanism of uracil and uridine. Mutant U^?, isolated from a culture of the parent strain, is resistant to 5-fluorouracil and is deficient in the uracil transport system. Mutant UR^?, isolated from a culture of the parent strain, is resistant to a low concentration of showdomycin and lacks the capacity to transport intact uridine. Mutant U^?UR^?isolated from a culture of mutant U^?, is resistant to a low concentration of showdomycin and is defective in both uracil and intact uridine transport processes. Mutant UR^?R^? was isolated from a culture of mutant UR^?, and is resistant to high concentration of showdomycin. This mutant is defective for transport of intact uridine and in addition lacks the transport system for the ribose moiety of uridine. Characteristics of uracil and uridine transport in parent and mutant cells demonstrate the existence of specific transport processes for uracil, intact uridine and the uracil and ribose moieties of uridine. Mutants U^? and UR^?, which are defective for uracil transport, lack uracil phosphoribosyltransferase activity and retain a small but significant capacity to transport uracil. The data support the conclusion that uracil is transported by two mechanisms, the major one of which requires uracil phosphoribosyltransferase activity, while the other process involves the transport of uracil as such. The characteristics of uridine transport in parent and mutant strains show that, in addition to transport as the intact nucleoside, uridine is rapidly cleaved to the uracil and ribose moieties. The latter is transported into the cell by a process which, in contrast to transport of intact uridine, does not require an energy source. The uracil moiety is released into the medium and is transported by the uracil transport system. Whole cells of the parent and mutant strains differ in their ability to cleave uridine even though cell-free extracts of all the strains have similar uridine phosphorylase activity. The data implicate a uridine cleavage enzyme in a group transport of the ribose moiety of uridine, a process which is nonfunctional in mutants which lack the capacity to transport the ribose moiety of uridine. A common transport component for this process and the transport of intact uridine is indicated by similarities in the inhibitory effects of heterologous nucleosides on these process.     

Lactose transport in Escherichia coli cells Dependence of kinetic parameters on the transmembrane electrical potential difference

We determine the kinetic parameters $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of lactose transport in Escherichia coli cells as a function of the electrical potential difference (Δψ) at pH 7.3 and $\text{Δ}\text{pH}\text{= 0}$. We report that transport occurs simultaneously via two components: a component which exhibits a high $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ (larger than 10 mM) and whose contribution is independent of Δψ, a component which exhibits a low $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ independent of Δψ (0.5 mM) but whose $\text{V}$ increases drastically with increasing Δψ. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon Δψ of $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ and $\text{V}$ of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49–63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H⁺/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s)) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H⁺/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H⁺/lactose stoichiometry different for active transport and facilitated diffusion.     

Parallel inductive kinetics of fluorescence and photoacoustic signal in dark-adapted thalli of Bryopsis maxima

The inductive kinetics of fluorescence and photoacoustic signal were measured simultaneously in dark-adapted thalli of the green coenocytic alga Bryopsis maxima. Under illumination with weak red light modulated at 60 Hz, the fluorescence yield varied, showing three maxima P, M₁ and M₂ almost immediately, 10 s and 6 min after the onset of the illumination, respectively (Yamagishi, A., Satoh, K. and Katoh, S. (1978) Plant Cell Physiol. 19, 17–25). The photoacoustic signal also showed inductive transients which parallel well those of the fluorescence up to the M₂ stage. After M₂, the photoacoustic signal remained at a constant level, while the emission yield gradually decreased. The first peak of the fluorescence induction and a corresponding peak of the photoacoustic transients were selectively eliminated by prior illumination or methyl viologen treatment of the dark-adapted thalli. The second peaks of the two induction curves were abolished by carbonylcyanide-m-chlorophenylhydrazone, whereas dicyclohexylcarbodiimide enhanced their peak heights and suppressed the subsequent decreases. The results indicate that the fluorescence yield is mainly determined by the redox state of the Photosystem II reaction center throughout the induction period except the last phase. Mechanisms underlying inductive transients of fluorescence are discussed in the light of the present findings.     

Reaction of cytochrome c in the electron-transport chain of Paracoccus denitrificans

The reaction of the cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of Paracoccus denitrificans cytoplasmic membranes with the endogenous cytochrome c of the membranes was studied, as well as its interaction with added exogenous cytochrome c from P. denitrificans or bovine heart. The polarographic method was employed, using N,N,N′,N′-tetramethyl-p-phenylenediamine plus ascorbate to reduce the cytochrome c. We found that overall electron transport can proceed maximally while the cytochrome c remains membrane bound; NADH or succinoxidase activities were not inhibited by the addition of substances which bind the P. denitrificans cytochrome c strongly. In contrast to our observations with the spectrophotometric method (Smith, L., Davies, H.C. and Nava, M.E. (1976) Biochemistry 15, 5827–5831), in the polarographic assays the membrane-bound oxidase reacts with about equal rapidity with exogenous bovine and P. denitrificans cytochromes c. The reaction of the oxidase with the endogenous cytochrome c proceeds at high rates and preferentially to that with exogenous cytochrome c; the reaction with the latter, but not the former is inhibited by positively charged poly(l-lysine). The cytochrome c and the oxidase appear to be very closely associated on the membrane.     

Sodium-dependent isoleucine transport in the methanogenic archaebacterium Methanococcus voltae

Methanococcus voltae possesses a Na⁺-dependent transport system for isoleucine which requires for optimum rates a CO₂/H₂ atmosphere. The K_m for the system is 4.5 μM with a V_max of 1.5 nmol·min^?1·mg dry wt^?1. Approximately 75% of the label can be released from the cell pool following short-term experiments with gradients of isoleucine reaching 100 (in/out). Transport is inhibited by ionophores and N-ethyl maleimide. Only valine and leucine effectively compete with isoleucine for transport.     

The inhibitory effect of N,N′-dicyclohexylcarbodiimide in active sugar uptake by Rhodotorula glutinis

Cells of the yeast Rhodotorula glutinis on treatment with N,N′-dicyclohexylcarbodiimide (DCCD) at a concentration of about 0.5 mM fail to accumulate d-xylose, cause efflux of accumulated sugar and do not exhibit H⁺/sugar symport. The results are interpreted as being due to depolarization of the membrane potential by DCCD.     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

Sodium-dependent uptake of taurine in encapsulated Staphylococcus aureus strain M

A novel uptake system for the unusual sulfonated amino acid taurine was discovered in the prokaryote, encapsulated Staphylococcus aureus strain M. This strain has been shown previously to contain taurine in its capsular polysaccharide. Taurine uptake by whole cells incubated in buffer showed a saturable dependency upon Na⁺ and taurine uptake was itself a saturable process, stimulated by glucose, and markedly affected by temperature. No evidence was found for the inducibility of taurine uptake. In the presence of 10 mM NaCl Lineweaver-Burk plots revealed a K_m of 42 μM and V_max of 4.6 nmol/min per mg dry weight for taurine uptake at 37°C. Increasing concentrations of Na⁺ decreased the K_m of the system and appeared to increase the V_max. Of various other cations tested only Li⁺ supported marked taurine uptake. Excess unlabelled taurine did not cause efflux of radioactivity taken up. Taurine was taken up into cold trichloroacetic acid-soluble material and did not chromatograph as taurine, indicating rapid metabolism during or closely following uptake. Taurine uptake appeared to occur via a highly specific system because amino acids representing the major known groups of amino acid transport systems in S. aureus did not inhibit taurine uptake, and uptake was only slightly diminished by the structurally closely related compounds hypotaurine and 3-amino-1-propane sulfonic acid. Sulfhydryl group reagents, electron transport inhibitors, an uncoupler and inhibitors of Na⁺-linked transport processes inhibited taurine uptake. A variety of other metabolic inhibitors had little effect on taurine uptake.     

The influence of uncouplers on facilitated diffusion of sorbose in Saccharomyces cerevisiae

Sorbose uptake in Saccharomyces cerevisiae, strain Delft 1, proceeds via mediated passive transport. In the cell sorbose is distributed in at least two compartments. Efflux studies showed that sorbose uptake in one of these compartments is not readily reversible. Uncouplers of oxidative phosphorylation inhibit both transport velocity and steady-state uptake level. It could be shown that these two effects are caused by different modes of action of the uncouplers. None of these two effects could be ascribed to changes of the electrochemical H⁺ gradient or of the intracellular pH. It is suggested that the inhibition of uptake velocity is caused by binding of the uncoupler to the sorbose translocator, thus lowering the transport activity. The uncoupler binding site is probably located at the intracellular fragment of the carrier. The second effect, reduction of the steady-state uptake level, is probably due to blocking of sorbose influx into the compartment that exhibits poor reversibility.     

d-Gluconate transport in Arthrobacter pyridinolis. Metabolic trapping of a protonated solute

d-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 5 μM}$). Uptake of d-gluconate was not mediated by a phosphoenolpyruvate: hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, ΔpH, of ?63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, $\text{ΔpH = 0}$. Addition of 8 μM d-gluconate significantly reduced the ΔpH. The transmembrane electrical potential, Δψ, which was ?87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. d-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the Δψ did not energize transport. Rather, the rate of d-gluconate uptake correlated with and appeared to be determined by the rate of d-gluconate metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both d-gluconate uptake and the metabolism of pre-accumulated d-gluconate; (b) the effects of cyanide and azide on d-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on d-gluconate metabolism; (c) extraction and chromatography of intracellular label from d-gluconate uptake revealed that accumulation of unaltered d-gluconate was negligible; (d) a series of mutant strains with lower d-gluconate kinase activities also exhibited low rates of d-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both d-gluconate kinase activity and wild type levels of uptake.     

The effects of uncoupler on the rates of cytochrome oxidation and reduction in the photosynthetic bacterium, Chromatium. Evidence for a possible cytochrome switching

The uncoupler, m-chlorocarbonyl cyanide phenylhydrazone (CCCP) added to anaerobic, dark-adapted whole cells of Chromatium vinosum is found to speed the reduction of cytochrome after oxidation by laser or by steady illumination and, subject to unknown factors, to slow the laser-induced oxidation. There is considerable evidence, including spectra and loss of low-temperature oxidizability that this results from a switch of the identity of the cytochrome oxidized from the low-potential cytochrome c-552 to the high-potential cytochrome c555. Redox control and/or control by conformational movements of the cytochromes, either being coupled to energy transduction in the cyclic system, are suggested as mechanisms for the switching. If the switching hypothesis is not accepted, the increased rate of reduction could alternatively be explained by postulating a phosphorylation site in the reduction pathway.     

Isolation of mutants of Escherichia coli uncoupled in oxidative phosphorylation using hypersensitivity to streptomycin

Mutants of Escherichia coli, harbouring the uncA401 or uncB402 alleles, were found to take up streptomycin more rapidly than the coupled parent strains. The increased rate of uptake results in greater sensitivity of the uncoupled strains, compared to the parent strains, to low concentrations of streptomycin. Studies with unc⁺ revertants showed that hypersensitivity to streptomycin is attributable to the mutation causing uncoupling. The uptake of streptomycin in an unc^? strain is abolished by addition of the chemical uncoupler carbonylcyanide m-chlorophenylhydrazone. The phenotype of hypersensitivity to streptomycin can be used as a selection procedure for the isolation of uncoupled strains. In an experiment reported here, nine out of 12 strains isolated as being sensitive to streptomycin (at 2.5 μg/ml), were found to be unable to grow on succinate as a sole source of carbon. Five of the nine Suc^? strains were found to be uncoupled in oxidative phosphorylation, and two of the five uncoupled strains lacked Mg²⁺-ATPase activity. The mutations causing uncoupling were cotransducible with the ilv genes.     

Inhibition of growth of Escherichia coli by lactose and other galactosides

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a β-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in ΔpH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-β-d-thiogalactoside.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases

The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II. The X-ray structures of both membrane-bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different. These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.