Ascorbate-phenazine Methosulfate Research Articles

Determination of the Absolute Number of Escherichia coli Membrane Vesicles That Catalyze Active Transport

Transport of vinylglycolate (2-hydroxy-3-butenoic acid) via the lactate transport system is the limiting step for covalent labeling of membrane vesicles prepared from E. coli ML 308-225. Thus, the rate and extent of vinylglycolate labeling is stimulated about 10-fold by ascorbate-phenazine methosulfate, and stimulation is abolished by 2,4-dinitrophenol and by phospholipase treatment, neither of which affect the rate of vinylglycolate oxidation. [(3)H]Vinylglycolate of high specific activity has been prepared, and vesicles have been labeled with this compound in the presence of ascorbate-phenazine methosulfate. Examination of these preparations by high resolution radioautography in the electron microscope demonstrates that virtually all of the vesicles are labeled. The experiments provide a strong indication that most, if not all, of the membrane vesicles in these preparations catalyze active transport.

The Effect of Sulfhydryl Inhibitors on Substrate Oxidation and Proline Transport with Membrane Preparations from Mycobacterium phlei

Abstract Substrate oxidation and amino acid transport were examined in the presence of sulfhydryl reagents with membrane preparations from Mycobacterium phlei. Oxidation of exogenously added β-NADH, succinate, ascorbate N, N, N', N'-tetramethyl-p-phenylenediamine, or ascorbate phenazine methosulfate was inhibited by organic mercurials. The organic mercurials inhibited succinate oxidation between cytochrome b and c. N-ethylmaleimide did not inhibit the oxidation of succinate, ascorbate N, N,N', N'-tetramethyl-p-phenylenediamine or ascorbate phenazine methosulfate, but did block NADH oxidation at the level of NADH dehydrogenase. The rate and steady state level of proline transport was lowered slightly by N-ethylmaleimide with all substrates. The organic mercurials inhibited the uptake of proline or induced proline efflux if added after transport steady state levels had been obtained. By altering the concentration of the mercurials, it was possible to obtain concentrations which did not inhibit substrate oxidation, but inhibited proline transport. The inhibition of transport was not as great if proline was preincubated with the membrane vesicles before the addition of inhibitor. Preincubation of the membrane preparations with the organic mercurials inhibited the nonenergized accumulation of proline; preincubation with proline afforded some protection from this inhibition. Thus, the organic mercurials appear to act at the level of a protein involved in transport. The ability to dissociate proline transport from substrate oxidation suggests that with membrane preparations from M. phlei there are a minimum of two distinct sulfhydryl moieties: one group is involved with proline transport and at least one group is involved with substrate oxidation.

Adenosine uptake by isolated membrane vesicles from Escherichia coli K-12

1. 1. Three different adenosine-uptake systems were found in isolated membrane vesicles of Escherichia coli K-12 cells: the first system was strongly inhibited by adenonine nucleosides, pyrimidine nucleosides and the antibiotic showdomycin but weakly inhibited by guanine nucleosides and inosine; the second system was strongly inhibited by adenine nucleosides, guanine nucleosides and inosine but weakly inhibited by pyrimidine nucleosides and showdomycin; and the third system was strongly inhibited by adenine. In isolated membrane vesicles of a showdomycin-resistant mutant (Shm r-001), the first system was no longer detectable but the second and the third systems were found to remain active. 2. 2. The adenosine uptake by these systems was stimulated by the artificial electron-donor system, ascorbate-phenazine methosulfate. The reduced phenazine methosulfate-stimulated uptake of adenosine by these systems was inhibited by sodium azide. 3. 3. Caffeine markedly inhibited the uptake of adenosine by the second system, but stimulated that of adenosine by the first system. 4. 4. Showdomycin strongly inhibited the uptake of adenosine by the first system but only slightly inhibited that of adenosine previously loaded membrane vesicles of Escherichia coli K-12 in the presence of adenine and caffeine. The reduced phenazine methosulfate-stimulated uptake of [ 14C]-showdomycin by membrane vesicles from Escherichia coli K-12 cells was strongly inhibited by adenosine and deoxycytidine but only slightly inhibited by guanosine. These results suggest that showdomycin is taken up by the first system of adenosine uptake in the membrane vesicles from Escherichia coli K-12 cells.

New procedure for the isolation of membrane vesicles of Bacillus subtilis and an electron microscopy study of their ultrastructure.

A rapid procedure for the isolation of membrane vesicles of Bacillus subtilis is described that minimizes the action of proteolytic enzymes, excreted by this organism, on the membrane proteins. The membrane vesicles obtained have, in addition to a low endogenous respiration rate, a low endogenous activity for transport of amino acids and carboxylic acids. In the presence of the electron donor, ascorbate-phenazine methosulfate, the transport activities for these compounds were comparable to the activities of intact cells. In addition, these activities were retained for a prolonged period of time. Electron microscopy examination of thin sections of the vesicles showed that the preparation consisted almost exclusively of membrane vesicles which were not contaminated with other cell components. The membrane vesicles, which are six to seven times smaller in diameter than protoplasts, often enclosed smaller vesicles. Freeze-etching of intact cells, protoplasts, and membrane vesicles showed that the orientation of the membrane of the vesicles was identical to the orientation of the plasma membrane in intact cells and protoplasts. This also held for the majority of the membranes of the enclosed vesicles, only 15% having the opposite orientation.

Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: XIII. VALINOMYCIN-INDUCED RUBIDIUM TRANSPORT

Valinomycin-induced uptake of rubidium by membrane vesicles prepared from Escherichia coli, Staphylococcus aureus, and Micrococcus denitrificans is analogous in nearly all respects to the transport of sugars or amino acids, or both, by these membrane vesicles. In E. coli membrane vesicles, concentrative rubidium uptake is stimulated maximally by d -lactate and by the artificial electron donor ascorbate-phenazine methosulfate, to a lesser extent by l -lactate, dl -α-hydroxybutyrate, succinate, and reduced diphosphopyridine nucleotide, and not at all by ATP, phosphoenolpyruvate, or a number of other metabolites. There is no direct relationship between the ability of various electron donors to stimulate rubidium uptake (ascorbate-phenazine methosulfate >> d -lactate >> l -lactate g dl -α-hydroxybutyrate g succinate g reduced diphosphopyridine nucleotide) and their rates of oxidation by membrane vesicles (ascorbate-phenazine methosulfate >> reduced diphosphopyridine nucleotide g succinate g d -lactate g l -lactate g dl -α-hydroxybutyrate). In the presence of d -lactate and valinomycin, the initial rate of rubidium uptake versus the rate of d -lactate oxidation yields a ratio of nearly 2:1. Initial rates of d -lactate-dependent rubidium uptake are saturable with respect to rubidium and valinomycin concentrations and exhibit the same temperature optimum at 50° as that observed for d -lactate oxidation. Steady state levels of rubidium accumulation vary with temperature and can be shifted readily from one steady state level to another by raising or lowering the temperature. Competitive uptake and displacement experiments indicate that rubidium and potassium are equivalent substrates for valinomycin-induced uptake. Rubidium uptake in the presence of valinomycin is markedly inhibited by anaerobiosis, by the electron transfer inhibitors oxamate, amytal, 2-heptyl-4-hydroxyquinoline-N-oxide and cyanide, and by p-chloromercuribenzenesulfonate, but not by arsenate or dicyclohexylcarbodiimide; inhibition by p-chloromercuribenzenesulfonate is reversed by dithiothreitol. Moreover, anaerobiosis, amytal, 2-heptyl-4-hydroxyquinoline-N-oxide, and cyanide produce rapid efflux of rubidium from the vesicles. Oxamate and p-chloromercuribenzenesulfonate, however, cause little or no efflux of rubidium accumulated in the presence of valinomycin, even though p-chloromercuribenzenesulfonate-treated vesicles catalyze exchange of intravesicular rubidium with external potassium at normal rates. These findings indicate that valinomycin does not simply catalyze the passive transfer of rubidium across the vesicle membrane. Valinomycin-induced active rubidium uptake is accompanied by the rapid efflux of intravesicular sodium against its concentration gradient. The vesicles are passively permeable to the lipophilic dibenzyldimethylammonium ion; however, this cation does not inhibit rubidium uptake or rubidium-induced sodium efflux and does not cause sodium efflux. Moreover, radioactive dibenzyldimethylammonium is not accumulated by membrane vesicles under conditions in which rubidium is actively transported. Valinomycin, in the presence of potassium, has no significant effect on the transient acidification of the medium observed upon addition of d -lactate to the vesicles. In addition, vesicles treated with Tween 40 such that they are rendered devoid of a diffusion barrier exhibit pH changes similar to those of untreated vesicles. These findings indicate that active rubidium uptake is an electrogenic process and that proton or potential gradients are not the primary driving force for respiration-linked active transport in isolated bacterial membrane vesicles.

Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: VIII. THE TRANSPORT OF AMINO ACIDS BY MEMBRANES PREPARED FROM ESCHERICHIA COLI

Abstract The studies presented in this paper demonstrate that the transport of 16 amino acids by membrane vesicles prepared from Escherichia coli ML 308–225 exhibits properties similar to those found previously for respiration-linked β-galactoside transport. Concentrative uptake of all 16 amino acids is stimulated maximally by d-lactate and by the artificial electron donor system ascorbate-phenazine methosulfate, and to a lesser extent by succinate, l-lactate, dl-α-hydroxybutyrate, and NADH. Competitive uptake and displacement studies indicate that the vesicles possess at least nine distinct amino acid uptake systems with the following substrate specificities: proline, lysine, glycine-alanine, serine-threonine, glutamic acid-aspartic acid, phenylalanine-tyrosine-tryptophan, histidine, leucine-isoleucine-valine, cysteine. In the presence of ascorbate-phenazine methosulfate, the initial rates of lysine, alanine, serine, glutamic acid, tyrosine, leucine, cysteine, and lactose transport by membrane vesicles are 42 to 102% of those exhibited by appropriately treated whole cells. Initial rates of d-lactate-dependent transport as a function of external amino acid concentration yield simple hyperbolic kinetics for all but four amino acids: histidine, leucine, isoleucine, and valine yield biphasic velocity curves. Apparent Km values determined for the amino acids are in general agreement with results reported for intact cells. Steady state levels of d-lactate-dependent amino acid accumulation vary with temperature. These levels represent a balance between rates of influx and efflux, which can be shifted readily from one steady state level to another by raising or lowering the temperature. Different amino acid transport systems exhibit different steady state temperature optima, ranging from 20°–35°. In contrast, temperature optima for the initial rate of uptake occur at the same temperature (45°) with nearly all of the transport systems, and are similar to the temperature optimum for d-lactic dehydrogenase. There is no relationship between rates of oxidation of electron donors by the membranes (NADH g d-lactate g succinate g dl-α-hydroxybutyrate g l-lactate) and the ability of these compounds to stimulate amino acid uptake. These results indicate that the site of energy coupling between respiration and amino acid transport lies between the primary dehydrogenase and cytochrome b1. Furthermore, the relative effectiveness of these electron donors in supporting uptake varies widely among the various transport systems. The d-lactate-coupled concentrative uptake of proline, lysine, alanine, threonine, aspartic acid, tyrosine, histidine, leucine, and cysteine is inhibited by anaerobiosis, by the electron transfer inhibitors cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, amytal, and oxamate, and by p-chloromercuribenzoate; inhibition by the latter compound is reversed by dithiothreitol. Furthermore, anaerobiosis, cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, and amytal cause rapid efflux of the same amino acids from the intravesicular pool. Oxamate and p-chloromercuribenzoate, however, produce little or no efflux of accumulated amino acids. Moreover, p-chloromercuribenzoate blocks tyrosine exchange and cyanide-induced tyrosine efflux; in each case inhibition is reversed by dithiothreitol. Finally, cyanide-induced efflux of proline exhibits a Km for intramembranal proline which is 350 times higher than the Km determined for d-lactate-dependent uptake of external proline, whereas the Vmax values are the same for both processes. The findings presented are consistent with the conceptual model suggested for d-lactic dehydrogenase-coupled β-galactoside transport.

Transport of Succinate in Escherichia coli: II. CHARACTERISTICS OF UPTAKE AND ENERGY COUPLING WITH TRANSPORT IN MEMBRANE PREPARATIONS

Abstract Membrane vesicles prepared from a strain of Escherichia coli which lacks membrane-bound succinate dehydrogenase and fumarate reductase activity are capable of accumulating succinate in the presence of d(-)lactate or reduced phenazine methosulfate as electron donors. The steady state, intramembranal concentration of succinate reaches 30- to 50-fold of the external concentration. Both the initial and the steady state rates are higher with ascorbate-phenazine methosulfate than with d(-)lactate. ATP, ADP, and various other metabolic intermediates do not stimulate succinate transport. Magnesium ions are required for maximum uptake. Monovalent cations, such as Na+, K+, or Li+ also stimulate transport to varying degrees. With ascorbate-phenazine methosulfate as electron donor the Michaelis constant for succinate is about 10 µm, and the inhibition constants for fumarate and l-malate are 12 µm and 22 µm, respectively. Both the ascorbate-phenazine methosulfate-dependent and d(-)lactate-dependent uptake of succinate are inhibited by energy poisons, uncouplers, and various other respiratory chain inhibitors. Compounds known to interfere with phosphorolytic and transphosphorylation reactions are without significant effect on transport. Amongst the thiol reagents, N-ethylmaleimide and showdomycin do not inhibit succinate uptake even at very high concentrations, but p-chloromercuribenzoate, Hg++ and Ag++ are very potent inhibitors. Both N-ethylmaleimide and p-chloromercuribenzoate, however, strongly inhibit d-lactate oxidation. From these observations, and from oxamate inhibition data, it is concluded that electrons are coupled to succinate transport directly at the initial step of lactate dehydrogenation. It is proposed that succinate uptake is mediated by alternate reduction and oxidation of a succinate carrier in membranes. Membranes prepared from strains carrying genes dct A or dct B are defective in the uptake of succinate. It thus appears that at least two genetic loci are involved in the formation of the succinate transport system in the membranes.

Mechanisms of Active Transport in Isolated Membrane Vesicles: IV. GALACTOSE TRANSPORT BY ISOLATED MEMBRANE VESICLES FROM ESCHERICHIA COLI

Abstract An inducible transport system for d-galactose is present in isolated membrane vesicles from Escherichia coli ML 3 and ML 35. Like the transport systems for β-galactosides and amino acids, galactose transport is coupled primarily to a membrane-bound d-lactic dehydrogenase. In addition to d-lactate, the transport system is markedly stimulated by ascorbate plus phenazine methosulfate and oxygen. Succinate, dl-α-hydroxybutyrate, and l-lactate partially replace d-lactate, but are less effective than either d-lactate or ascorbate-phenazine methosulfate. ATP and P-enolpyruvate, in addition to a number of other metabolites and cofactors, do not stimulate galactose transport by the vesicles. transport by isolated membrane vesicles requires oxygen, and is blocked by electron transfer inhibitors and by other general metabolic inhibitors. However, transport is not significantly inhibited by high concentrations of arsenate or oligomycin. Galactose binding protein is totally absent from the vesicles, as is a high affinity galactose transport system present in whole cells.

Active Transport in Isolated Bacterial Membrane Vesicles: V. THE TRANSPORT OF AMINO ACIDS BY MEMBRANE VESICLES PREPARED FROM STAPHYLOCOCCUS AUREUS

Abstract Concentrative uptake of 16 amino acids by membrane vesicles isolated from Staphylococcus aureus is stimulated 3 to 100 times by the conversion of l-α-glycerol phosphate to dihydroxyacetone phosphate. With the exception of ascorbate-phenazine methosulfate, d-lactate, phosphoenolpyruvate, ATP, and a number of other metabolites and cofactors do not replace α-glycerol phosphate. Amino acid transport by these membrane preparations in the presence of α-glycerol phosphate requires oxygen, and is blocked by potassium cyanide, sodium azide, and dinitrophenol; however, uptake is not significantly inhibited by high concentrations of arsenate. Vesicles contain insignificant amounts of ATP; and the level of ATP is not increased by incubation in the presence of α-glycerol phosphate or NADH. The membrane-bound α-glycerol phosphate dehydrogenase is coupled to oxygen via a cytochrome system also present in the vesicle membrane, and spectrophotometric evidence shows that α-glycerol phosphate dehydrogenase, NADH dehydrogenase, l-lactic dehydrogenase, and succinic dehydrogenase all utilize the same cytochrome system. There is no relationship between rates of oxidation of electron donors by the respiratory chain (NADH g α-glycerol phosphate >> l-lactatesuccinate) and the ability of these compounds to stimulate amino acid transport. N-Ethylmaleimide and p-hydroxymercuribenzoate inhibit amino acid transport and α-glycerol phosphate oxidation. However, N-ethyl-maleimide does not significantly inhibit α-glycerol phosphate dehydrogenase with dichlorophenolindophenol as an artificial acceptor, nor does it inhibit oxygen utilization in the presence of NADH. These findings indicate that the site of coupling of α-glycerol phosphate dehydrogenase to amino acid transport lies between the primary dehydrogenase and the cytochrome chain. It is concluded that amino acid transport in S. aureus is catalyzed by mechanisms similar to those found in Escherichia coli with the exception that α-glycerol phosphate, rather than d-lactate, is the primary electron donor.

Mechanisms of Active Transport in Isolated Membrane Vesicles: III. THE COUPLING OF REDUCED PHENAZINE METHOSULFATE TO THE CONCENTRATIVE UPTAKE OF β-GALACTOSIDES AND AMINO ACIDS

Abstract An artificial electron donor system—ascorbate and phenazine methosulfate—markedly stimulates β-galactoside transport in isolated membrane vesicles from Escherichia coli ML 308-225. Maximal rates of transport are dependent on the presence of both ascorbate and phenazine methosulfate and also oxygen. Moreover, in the presence of phenazine methosulfate, reduced nicotinamide adenine dinucleotide stimulates the initial rate of lactose transport. The effect of ascorbate-phenazine methosulfate is inhibited by removal of oxygen or potassium cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, p-chloromercuribenzoate, or sodium amytal. Oxamic acid has no significant effect. Ascorbate-phenazine methosulfate reduces the respiratory chain of the vesicles below the potential level of cytochrome b1, but above approximately 80% of the membrane-bound flavoproteins. In the presence of ascorbate-phenazine methosulfate, membranes prepared from Escherichia coli, Salmonella typhimurium, Pseudomonas putida, Proteus mirabilis, Bacillus megaterium, and Bacillus subtilis catalyze the concentrative uptake of proline; membranes prepared from Staphylococcus aureus catalyze the concentrative uptake of lysine; and membranes prepared from Micrococcus dentrificans catalyze the concentrative uptake of glutamine.