Potassium Transport and the Relationship Between Intracellular Potassium Concentration and Amino Acid Uptake by Cells of a Marine Pseudomonad

Transport of K(+) by K(+)-depleted cells of marine pseudomonad B-16 (ATCC 19855) exhibited saturation kinetics. Rb(+) inhibited both K(+) transport and the K(+)-dependent transport of alpha-aminoisobutyric acid (AIB) into K(+)-depleted cells of the organism in proportion to the concentration of Rb(+) in the suspending medium. Inhibition of the K(+)-dependent uptake of AIB into K(+)-depleted cells by Rb(+) could be overcome by increasing the concentration of K(+) in the medium. When AIB and K(+) were added simultaneously to a suspension of K(+)-depleted cells, the uptake of K(+) occurred immediately and rapidly, whereas the accumulation of AIB occurred only after a lag. The initial uptake rate of AIB was directly proportional to the intracellular K(+) concentration. The intracellular concentration of K(+) and AIB at their steady-state levels increased to a maximum as the Na(+) concentration in the suspending medium was increased. At Na(+) concentrations between 0.2 and 0.3 M, the molar ratio of K(+) to AIB at their intracellular steady-state concentrations was constant at 1.6. At external Na(+) concentrations less than 0.2 M, the cells maintained a relatively higher K(+) intracellular steady-state level than AIB.     

Nature of the specificity of alcohol coupling to L-alanine transport into isolated membrane vesicles of a marine pseudomonad

Ethanol stimulated the uptake of l-alanine into isolated membrane vesicles of a marine pseudomonad at a rate and to an extent comparable with that obtained with reduced nicotinamide adenine dinucleotide (NADH) or the artificial electron donor ascorbate-N, N, N', N'-tetramethyl-p-phenylenediamine (ascorbate-TMPD). Methanol and branched-chain alcohols had little or no capacity to energize transport. No quantitative relationship was found between the ability of a compound to induce oxygen uptake and to energize transport, since with ethanol initial rates of oxygen uptake were approximately 4% of that obtained with NADH or ascorbate-TMPD. Cytochrome analysis revealed that NADH and ethanol reduced cytochromes b and c, whereas ascorbate-TMPD coupled primarily at the level of cytochrome c. Approximately 25% of the cytochromes reduced by dithionite were reducible by ethanol. Ethanol reduction of both cytochromes b and c was prevented by 2-heptyl-4-hydroxyquinoline-N-oxide, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. The ethanol- and NADH-energized transport systems for l-alanine were subject to quantitatively similar inhibition by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, 2, 4-dinitrophenol, and the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. In contrast, for ascorbate-TMPD-driven transport, only cyanide and 2, 4-dinitrophenol remained fully effective as inhibitors, p-chloromercuribenzoate was only half as effective, and the other compounds stimulated transport. Inhibition of ethanol oxidation strikingly paralleled the inhibition of ethanol-driven transport for each of the inhibitors, including 2, 4-dinitrophenol. Marked differences between inhibition of oxygen uptake and inhibition of transport were observed when NADH or ascorbate-TMPD were the electron donors. The data indicate that only a small proportion of the respiratory chain complexes in the membrane vesicles are involved in transport and these are efficiently coupled to ethanol oxidation. The results also suggest that when 2, 4-dinitrophenol inhibits transport it is not acting as an uncoupling agent.     

Relationship between ion requirements for respiration and membrane transport in a marine bacterium.

Intact cells of the marine bacterium Alteromonas haloplanktis 214 oxidized NADH, added to the suspending medium, by a process which was stimulated by Na+ or Li+ but not K+. Toluene-treated cells oxidized NADH at three times the rate of untreated cells by a mechanism activated by Na+ but not by Li+ or K+. In the latter reaction, K+ spared the requirement for Na+. Intact cells of A. haloplanktis oxidized ethanol by a mechanism stimulated by either Na+ or Li+. The uptake of alpha-aminoisobutyric acid by intact cells of A. haloplanktis in the presence of either NADH or ethanol as an oxidizable substrate required Na+, and neither Li+ nor K+ could replace it. The results indicate that exogenous and endogenous NADH and ethanol are oxidized by A. haloplanktis by processes distinguishable from one another by their requirements for alkali metal ions and from the ion requirements for membrane transport. Intact cells of Vibrio natriegens and Photobacterium phosphoreum oxidized NADH, added externally, by an Na+-activated process, and intact cells of Vibrio fischeri oxidized NADH, added externally, by a K+-activated process. Toluene treatment caused the cells of all three organisms to oxidize NADH at much faster rates than untreated cells by mechanisms which were activated by Na+ and spared by K+.     

Amino acid transport in isolated rat thymocytes. Effects of divalent cations and ethanol.

In dispersed rat thymocytes neither basal alpha-aminoisobutyric acid influx nor influx stimulated by insulin, prostaglandin theophylline, or butyryl adenosine 3':5'-monophosphate (cyclic AMP) depended on extracellular calcium or magnesium. The divalent cation ionophore A23187 inhibited both basal and stimulated alpha-aminoisobutyric acid influx. The extent to which influx was inhibited depended on ionophore concentration, extracellular calcium concentration, and time but did not depend on extracellular magnesium. Significant inhibition could be detected at an ionophore concentration of 1 muM and maximal inhibition occurred with 6 muM A23187. A23187 increased cellular uptake of calcium and there was good agred calcium uptake and that for ionophore inhibition of alpha-aminoisobutyric acid influx. Incubating cells with A23187 and then adding ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid completely reversed ionophore-stimulated cellular calcum uptake but did not reverse inhibition of alpha-aminoisobutyric acid influx. Thus, A23187 produces irreversible inhibition of alpha-aminoisobutyric acid transport in dispersed rat thymocytes. Ethanol abolished insulin-stimulated alpha-aminoisobutyric acid influx but did not alter basal influx or that stimulated by prostaglandin E1, theophylline, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate. Inhibition could be detected with 0.2% (v/v) ethanol and insulin-stimulated alpha-aminoisobutyric influx was abolished with 1% ethanol. The effect of ethanol occurred immediately and could be reversed completely. This ability of ethanol to inhibit selectively insulin-stimulated alpha-aminoisobutyric acid influx indicates that the mechanism through which insulin stimulates alpha-aminoisobutyric acid influx is functionally distinct from the stimulation produced by cyclic AMP.     

T-2 toxin inhibits mitochondrial function in yeast

T-2 toxin inhibits oxygen consumption of whole cells and purified mitochondria of Saccharomyces cerevisiae. Inhibition of mitochondrial respiration is not relieved by 2, 4-dinitrophenol, indicating that T-2 toxin inhibits mitochondrial function at the level of the electron transport chain. T-2 toxin inhibition of state 3 respiration (with succinate) is overcome by N, N, N', N'-tetramethyl-p-phenylenediamine, indicating inhibition of site II of the electron transport chain. T-2 toxin inhibits mitochondrial succinate dehydrogenase activity and increases mitochondrial NADH dehydrogenase activity.     

Optical spectra and kinetics of reactions of prostaglandin H synthase: effects of the substrates 13-hydroperoxyoctadeca-9,11-dienoic acid, arachidonic acid, N,N,N',N'-tetramethyl-p-phenylenediamine, and phenol and of the nonsteroidal anti-inflammatory drugs aspirin, indomethacin, phenylbutazone, and bromfenac

A combination of cyclooxygenase activity assays, rapid spectrophotometry and pre-steady-state, steady-state, and transient-state kinetics is used to characterize further the properties of prostaglandin H synthase. 13-Hydroperoxyoctadeca-9-11-dienoic acid is used as oxidizing substrate and the effects of the following compounds are examined: arachidonic acid, N,N,N',N'-tetramethyl-p-phenylenediamine, phenol, diethyldithiocarbamate, and the nonsteroidal anti-inflammatory drugs aspirin, indomethacin, phenylbutazone, and Bromfenac. The order of reactivity of four of these substrates, predominantly with compound II of prostaglandin H synthase, is N,N,N',N'-tetramethyl-p-phenylenediamine greater than phenol greater than indomethacin approximately phenylbutazone. Aspirin exhibits no effect. Arachidonic acid causes inactivation. Diethyldithiocarbamate acts as a reducing substrate for the oxidized forms of prostaglandin H synthase. Bromfenac appears to act both as a protective agent and inhibitor.     

The respiratory system of Chromobacterium violaceum grown under conditions of high and low cyanide evolution.

The particulate fraction of disrupted Chromobacterium violaceum grown under cyanide-evolving conditions was unable to oxidize ascorbate plus N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD), but oxidized NADH and succinate by a linear respiratory pathway which was very resistant to inhibition by cyanide. When the bacteria were grown under conditions where little cyanide evolution occurred, particulate fractions developed the ability to oxidize ascorbate-TMPD by a pathway highly sensitive to cyanide inhibition; respiratory activity with NADH and succinate proceeded via both the cyanide-sensitive and-resistant pathways. Studies with respiratory inhibitors, and the cytochrome compositions of the fractions derived from cultures grown under both conditions, are presented. A soluble, carbon monoxide-binding cytochrome c was found, and this appears similar to those found recently in Beneckea natiegens, methylotrophic bacteria and the marine pseudomonad B16.     

The H(+)-motive and Na(+)-motive respiratory chains in Bacillus FTU subcellular vesicles.

Respiration-dependent pumping of Na+ and H+ into the inside-out subcellular vesicles of alkalotolerant and halotolerant Bacillus FTU grown at alkaline pH was studied. The vesicles were shown to be competent in Na+ and H+ transport coupled to ascorbate oxidation via N,N,N',N'-tetramethyl-p-phenylenediamine or diaminodurene. The uphill Na+ uptake is strongly stimulated by either protonophores or valinomycin, whereas H+ uptake is stimulated by valinomycin and completely inhibited by protonophores. The salt of a penetrating weak base and of the penetrating weak acid, diethylammonium acetate, potentiates the stimulating effect of protonophores on Na+ uptake and abolishes H+ uptake. Na+ transport, supported by ascorbate oxidation, is resistant to 2-heptyl-4-hydroxyquinoline N-oxide, but sensitive to Ag+ and Na+ ionophore, N,N'-dibenzyl-N,N'-diphenyl-1,2-phenylenediacetamide. Micromolar concentrations of cyanide specifically inhibit the H+ uptake but does not affect Na+ uptake. These cyanide concentrations are shown to cause 70% inhibition of respiration, complete reduction of alpha-type cytochromes and partial reduction of c/b-type cytochromes. To inhibit the remaining respiratory activity and Na/ uptake, approximately 100-fold higher cyanide concentrations are necessary. High cyanide concentrations cause some additional increase in absorbance in the region of cytochromes c and/or b. In the presence of a high cyanide concentration, Na+ uptake can be supported by NADH oxidation by fumarate. This Na+ transport is stimulated by protonophores and diethylammonium acetate, being sensitive to very low concentrations of 2-heptyl-4-hydroxyquinoline N-oxide and Ag+. The NADH-fumarate reductase reaction is also found to be competent in H+ uptake, which is inhibited by protonophores and by much higher 2-heptyl-4-hydroxyquinoline N-oxide concentrations, and is resistant to Ag+. It is inferred that Bacillus FTU possesses two respiratory chains: the H(+)-motive and the Na(+)-motive, which strongly differ in their inhibitor sensitivities. Each chain comprises at least two energy-coupling sites which are localized in their initial and terminal segments. It has been indicated that common redox carrier(s) are present in the two chains.     

Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation.

Cultured cells of a Rhizobium phaseoli wild-type strain (CE2) possess b-type and c-type cytochromes and two terminal oxidases: cytochromes o and aa3. Cytochrome aa3 was partially expressed when CE2 cells were grown on minimal medium, during symbiosis, and in well-aerated liquid cultures in a complex medium (PY2). Two cytochrome mutants of R. phaseoli were obtained and characterized. A Tn5-mob-induced mutant, CFN4201, expressed diminished amounts of b-type and c-type cytochromes, showed an enhanced expression of cytochrome oxidases, and had reduced levels of N,N,N',N'-tetramethyl-p-phenylenediamine, succinate, and NADH oxidase activities. Nodules formed by this strain had no N2 fixation activity. The other mutant, CFN4205, which was isolated by nitrosoguanidine mutagenesis, had reduced levels of cytochrome o and higher succinate oxidase activity but similar NADH and N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activities when compared with the wild-type strain. Strain CFN4205 expressed a fourfold-higher cytochrome aa3 content when cultured on minimal and complex media and had twofold-higher cytochrome aa3 levels during symbiosis when compared with the wild-type strain. Nodules formed by strain CFN4205 fixed 33% more N2 than did nodules formed by the wild-type strain, as judged by the total nitrogen content found in plants nodulated by these strains. Finally, low-temperature photodissociation spectra of whole cells from strains CE2 and CFN4205 reveal cytochromes o and aa3. Both cytochromes react with O2 at -180 degrees C to give a light-insensitive compound. These experiments identify cytochromes o and aa3 as functional terminal oxidases in R. phaseoli.     

Glucagon treatment stimulates the metabolism of hepatic submitochondrial particles

Hepatic submitochondrial particles, prepared at neutral pH from rats pretreated with glucagon, exhibited stimulated rates of State 3 and uncoupled respiration when succinate or NADH were the substrates, but not when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine were employed. Measurements of 8-anilino-1-naphthalenesulfonic acid fluorescence in the particles indicated that glucagon treatment resulted in a stimulation of energization supported by succinate respiration or ATP hydrolysis. Similarly, the energy-linked pyridine nucleotide transhydrogenase and reverse electron flow reactions driven by succinate oxidation or ATP were also stimulated. The results indicate that mitochondrial substrate transport is not the prime locus of glucagon action. It is suggested that the increased level of energization in particles prepared from glucagon-treated rats is a reflection of a stimulation of the respiratory chain, possibly between cytochromes b and c, and the ATP-forming reactions.     

Stability and comparative transport capacity of cells, mureinoplasts, and true protoplasts of a gram-negative bacterium

The outer layers of the cell envelope of a pseudomonad of marine origin were removed by washing the cells in 0.5 m NaCl followed by suspension in 0.5 m sucrose. The term mureinoplast has been suggested for the rod-shaped forms which resulted from this treatment. As previously established, these forms lacked the outer cell wall layers but still retained a rigid peptidoglycan structure. Mureinoplasts remained stable if suspended in a balanced salt solution containing 0.3 m NaCl, 0.05 m MgSO(4), and 0.01 m KCl but, unlike whole cells, lost ultraviolet (UV)-absorbing material if suspended in 0.5 m NaCl or 0.05 m MgCl(2). Sucrose added to the balanced salt solution also enhanced the loss of UV-absorbing material. Addition of lysozyme to suspensions of mureinoplasts in the balanced salt solution produced spherical forms which, by electron microscopy and the analysis of residual cell wall material, appeared to be true protoplasts. Only undamaged mureinoplasts, as judged by their capacity to fully retain alpha-aminoisobutyric acid, were capable of being converted to protoplasts. Protoplasts and undamaged mureinoplasts retained 100% transport capacity when compared to an equal number of whole cells. The Na(+) requirement for transport of alpha-aminoisobutyric acid and the sparing action of Li(+) on this Na(+) requirement were the same for both protoplasts and whole cells. These observations indicate that, in this gram-negative bacterium, the cell wall does not participate in the transport process though it does stabilize the cytoplasmic membrane against changes in porosity produced by unbalanced salt solutions. The results also indicate that the requirements for Na(+) for transport and for the retention of intracellular solutes are manifested at the level of the cytoplasmic membrane.     

Comparative transport activity of intact cells, membrane vesicles, and mesosomes of Bacillus licheniformis

Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His(-). Lithium ion had a slight capacity to replace Na(+) in this capacity, but K(+) was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K(2(5)) or K(2(10)). Sodium ion stimulated transport of glutamic acid by membrane vesicles.     

Transport and retention of K+ and other metabolites in a marine pseudomonad and their relation to the mechanism of optical effects

Suspensions of cells of a marine pseudomonad washed with 0.05 m MgSO(4) showed an immediate increase in optical density (first-phase optical change) when the salt concentration of the suspending medium was increased; a subsequent slow decrease in optical density (second-phase optical change) occurred if K(+) was present. The rate of the second-phase change was similar to the rate of uptake of (42)K(+) by the cells. Glutamate increased the rate and extent of the second-phase change and produced a parallel increase in the rate and extent of uptake of (42)K(+). Citrate increased the extent of the second-phase change in cells adapted to oxidize citrate but not in unadapted cells. Adapted, but not unadapted, cells accumulated (14)C-citrate. The nonmetabolizable alpha-aminoisobutyric acid (AIB) also increased the extent of the second-phase change under conditions leading to the uptake of (14)C-AIB by the cells. Cells maintained in a salt solution optimal for the retention of intracellular solutes were found to contain 0.184 m K(+). In the same salt solution, cells preloaded with (42)K(+) retained the isotope, but they lost it rapidly when suspended in 0.05 m MgSO(4). The second-phase changes can be accounted for by the energy-dependent accumulation in an osmotically active form of K(+) and other metabolites by cells depleted of intracellular solutes.     

Further studies on lipid-peroxide formation in isolated hepatocytes.

Lipid peroxide formation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30-60 min, and the response was dose-related with iron concentrations ranging from 19 to 187 muM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The addition in vitro of N,N,N',N'-tetramethyl-p-phenylenediamine, menadione or p-benzoquinone inhibited the iron-induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N',N'-tetramethyl-p-phenylenediamine (100 muM). The attenuation of the iron-induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells. Cumene hydroperoxide-induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 muM to the liver cell incubate. With concentrations below 150 muM, a lag phase was present which seemed to be glutathione-dependent. It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPH via the NADPH-cytochrome P-450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron-apoferritin complex formation.     

Transport of amino acids into the oestrogen-primed uterus. Enhancement of the uptake by a preliminary incubation

1. Preincubation of the immature rat uterus under physiological conditions was found to increase its subsequent ability to transport alpha-aminoisobutyric acid, l-proline, l-alanine and 1-aminocyclopentanecarboxylic acid. Uptakes of l-valine, l-phenylalanine and l-leucine were not affected. With alpha-aminoisobutyric acid, a doubling of the uptake could be obtained after a 3-5h preincubation period. Uteri from oestradiol-primed rats gave increases similar to those found in tissues from untreated animals. In both cases the preincubation increased the V(max.) of alpha-aminoisobutyric acid uptake but did not affect the K(m). 2. The conditions during the preincubation period determined the increase in subsequent uptake of alpha-aminoisobutyric acid. No increase in uptake was found if the preincubation was carried out at 1 degrees C, in the presence of cyanide or dinitrophenol, under anaerobiosis or with a concentration of puromycin that depressed incorporation of l-leucine into protein by 95%. The puromycin was also shown to prevent the increase in V(max.) normally found after the preincubation period. In addition, no increase was found if Na(+) was omitted from the preincubation medium. Other inorganic ions had smaller effects. 3. The uptake of alpha-aminoisobutyric acid by uteri before and after a preincubation period showed the same general patterns of sensitivity to competitive inhibitors, K(+), pH, temperature and 2,4-dinitrophenol. 4. The results suggest that the preincubation leads to an increase in a protein component of the ;A system' for amino acid transport in the uterus, and that metabolic energy is required for the reactions involved.     

Immunological characterization of an Escherichia coli strain which is lacking cytochrome d.

The isolated membranes from an Escherichia coli mutant strain which lacks spectroscopically detectable levels of cytochromes d, a1, and b558 also have abnormally low levels of N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity. In this paper, it is shown that the material previously identified as the N,N,N',N'-tetramethyl-p-phenylenediamine oxidase is, in fact, the two-subunit cytochrome d complex. Antisera directed against the native cytochrome d complex as well as against each of two subunits apparent on sodium dodecyl sulfate-polyacrylamide gels were used to show that the mutant strain lacks both subunits of the cytochrome d complex. Introduction of F-prime F152 into the mutant strain restored the two subunits along with the spectroscopic and enzymatic activity associated with the cytochrome d complex.     

Relation between reduced nicotinamide adenine dinucleotide oxidation and amino acid transport in membrane vesicles from Bacillus subtilis.

The rate of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane vesicles from Bacillus subtilis W23 increases three- to fourfold during logarithmic growth, reaching maximal levels in early stationary phase. Initial rates of L-proline, L-alanine, and L-glutamate transport energized by NADH closely parallel the increase in NADH oxidation. In vesicles prepared at different stages of growth, a constant number of NADH molecules varying from 150 to 260 have to be oxidized to transport one molecule of amino acid. Membrane vesicles from B. subtilis aroD (strain RB163), a mutant defective in menaquinone synthesis, do not transport amino acids in the presence of NADH. Ascorbate plus phenazine methosulfate, however, energizes amino acid transport equally well as in vesicles of B. subtilis W23. NADH oxidation and NADH-driven amino acid transport can be restored instantaneously by the addition of menadione (vitamin K3).     

Sodium and Potassium Requirements for Active Transport of Glutamate by Escherichia coli K-12

Active transport of glutamate by Escherichia coli K-12 requires both Na(+) and K(+) ions. Increasing the concentration of Na(+) in the medium results in a decrease in the K(m) of the uptake system for glutamate; the capacity is not affected. Glutamate uptake by untreated cells is not stimulated by K(+). K(+)-depleted cells show a greatly reduced capacity for glutamate uptake. Preincubation of such cells in the presence of K(+) fully restores their capacity for glutamate uptake when Na(+) ions are also present in the uptake medium. Addition of either K(+) or Na(+) alone restores glutamate uptake to only about 20% of its maximum capacity in the presence of both cations. Changes in K(+) concentration affect the capacity for glutamate uptake but have no effect on the K(m) of the glutamate transport system. Ouabain does not inhibit the (Na(+)-K(+))-stimulated glutamate uptake by intact cells or spheroplasts of E. coli K-12.     

Respiratory physiology and energy conservation efficiency of Campylobacter jejuni

A study of the electron transport chain of the human intestinal pathogen Campylobacter jejuni revealed a rich complement of b- and c-type cytochromes. Two c-type cytochromes were partially purified: one, possibly an oxidase, bound carbon monoxide whereas the other, of high potential was unreactive with carbon monoxide. Respiratory activities determined with membrane vesicles were 50- to 100-fold higher with formate and hydrogen than with succinate, lactate, malate, or NADH as substrates. Evidence for three terminal respiratory components was obtained from respiratory kinetic studies employing cyanide, and the following Ki values for cyanide were determined from Dixon plots: ascorbate + reduced N,N,N', N'-tetramethyl-p-phenylenediamine, K1 + 3.5 muM; malate, K1 = 55 muM; and hydrogen, K1 = 4.5 muM. Two oxidases (K1 = 90 muM, 4.5 mM) participated in the oxidation of succinate, lactate, and formate. Except with formate, 37 muM HQNO inhibited respiration by approximately 50%. Carbon monoxide had little inhibitory effect on respiration except under low oxygen tension (less than 10% air saturation). The stoichiometry of respiratory-driven proton translocation (H+/O) determined with whole cells was approximately 2 for all substrates examined except hydrogen (H+/) = 3.7) and formate (H+/O = 2.5). The higher stoichiometries observed with hydrogen and formate are consistent with their respective dehydrogenase being located on the periplasmic face of the cytoplasmic membrane. The results of this study suggest that the oxidation of hydrogen and formate probably serves as the major sources of energy for growth.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.