The involvement of the electron transport chain in uptake of testosterone by membrane vesicles of Pseudomonas testosteroni

Uptake of testosterone by membrane vesicles of Pseudomonas testosteroni is inhibited when examined under anaerobic conditions and in the presence of the electron transport chain inhibitors, sodium amytal and antimycin A. These results indicated the involvement of the electron transport chain in steroid uptake by membrane vesicles.     

Testosterone uptake by membrane vesicles of Pseudomonas testosteroni

Uptake of testosterone was demonstrated in membrane vesicles prepared from Pseudomonas testosteroni grown on testosterone. In contrast, membrane vesicles from uninduced cultures revealed no significant transport activity for steroids. The K_m of the reaction was 2 · 10⁻⁶M and the V 28.5 nmoles/min per mg protein. Steroid uptake was maximal within the pH range of 8 to 9 and at incubation temperatures between 30 and 37 °C. Transport of steroid was dependent upon NAD⁺ and was reduced by NADH, dinitrophenol, and inhibitors of electron transport, such as N_3⁻ · CN⁻ and amytal. The intravesicular steroid concentration was approx. 800 times the steroid concentration present in the medium at the start of the incubation.     

Effects of colicin A and staphylococcin 1580 on amino acid uptake into membrane vesicles of Escherichia coli and Staphylococcus aureus

Staphylococcin 1580 increased the relative amount of diphosphatidylglycerol and decreased the amount of phosphatidylglycerol in cells of Staphlococcus aureus, while the amounts of lysylphosphatidylglycerol, phosphatidic acid and total phospholipid remained constant.Treatment of cells of Escherichia coli and S. aureus with colicin A and staphylococcin 1580, respectively, did not affect proton impermeability but subsequent addition of carbonylcyanide-m-chlorophenylhydrazone resulted in a rapid influx of protons into the cells.Bacteriocin-resistant and -tolerant mutants of E. coli and S. aureus were isolated. The bacteriocins caused leakage of amino acids preaccumulated into membrane vesicles of resistant mutants and had no significant effect on membrane vesicles of tolerant mutants.The uptake of amino acids into membrane vesicles was inhibited by both bacteriocins, irrespective of the electron donors applied. The bacteriocin inhibition was noncompetitive. The bacteriocins did not affect oxygen consumption and dehydrogenases in membrane vesicles.Both bacteriocins suppressed the decrease in the fluorescence of 1-anilino-8-naphthalene sulfonate caused by d-lactate or α-glycerol phosphate when added to membrane vesicles.It is concluded that the bacteriocins uncouple the transport function from the electron transport system.     

The effect of carbonyl cyanide m-chlorophenylhydrazone on steroid transport in membrane vesicles of Pseudomonas testosteroni

The uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) was an effective inhibitor of steroid transport in membrane vesicles of Pseudomonas testosteroni between 10 microM and 1 microM CCCP. At these concentrations the inhibition of steroid transport was not due to an inhibition of the 3 beta and 17 beta-hydroxysteroid dehydrogenase enzyme. CCCP also affected testosterone-dependent oxygen consumption at concentrations up to 100 microM and inhibited respiration at 0.5 and 1 microM. The effect of CCCP on testosterone-dependent oxygen consumption indicated that CCCP was acting as an uncoupler. The concurrent inhibition of testosterone transport and stimulation of testosterone-dependent oxygen consumption at 10-100 microM CCCP supported the conclusion that transport and metabolism were tightly coupled processes. When membrane vesicles were pre-incubated with CCCP for 15 min, CCCP did inhibit transport and the 3 beta and 17 beta-hydroxysteroid dehydrogenase activity. However, both transport and enzyme inhibition could be prevented by the addition of NAD+ to the incubation mixture. This indicated that CCCP exhibits the properties of a sulfhydryl reagent under pre-incubated conditions.     

Transmembrane Electron Transport in Plasma Membrane Vesicles Loaded with an NADH-Generating System or Ascorbate

Sugar beet (Beta vulgaris L.) leaf plasma membrane vesicles were loaded with an NADH-generating system (or with ascorbate) and were tested spectrophotometrically for their ability to reduce external, membrane-impermeable electron acceptors. Either alcohol dehydrogenase plus NAD⁺ or 100 millimolar ascorbate was included in the homogenization medium, and right-side-out (apoplastic side-out) plasma membrane vesicles were subsequently prepared using two-phase partitioning. Addition of ethanol to plasma membrane vesicles loaded with the NADH-generating system led to a production of NADH inside the vesicles which could be recorded at 340 nanometers. This system was able to reduce 2,6-dichlorophenolindophenol-3′-sulfonate (DCIP-sulfonate), a strongly hydrophilic electron acceptor. The reduction of DCIP-sulfonate was stimulated severalfold by the K⁺ ionophore valinomycin, included to abolish membrane potential (outside negative) generated by electrogenic transmembrane electron flow. Fe³⁺-chelates, such as ferricyanide and ferric citrate, as well as cytochrome c, were not reduced by vesicles loaded with the NADH-generating system. In contrast, right-side-out plasma membrane vesicles loaded with ascorbate supported the reduction of both ferric citrate and DCIP-sulfonate, suggesting that ascorbate also may serve as electron donor for transplasma membrane electron transport. Differences in substrate specificity and inhibitor sensitivity indicate that the electrons from ascorbate and NADH were channelled to external acceptors via different electron transport chains. Transplasma membrane electron transport constituted only about 10% of total plasma membrane electron transport activity, but should still be sufficient to be of physiological significance in, e.g. reduction of Fe³⁺ to Fe²⁺ for uptake.     

Distribution and effect of the weak acids 5,5-dimethyl-2,4-oxazolidinedione (DMO) and 2,4-dinitrophenol (DNP) in membrane vesicles of Micrococcus denitrificans.

The effects of 5,5-dimethyl-2,4-oxazolidinedione (DMO) and 2,4-dinitrophenol (DNP) on membrane vesicles of Micrococcus denitrificans were compared. DMO did not affect the ability of these vesicles to accumulate glycine in the presence of the substrate l-lactate. Both glycine transport and l-lactate oxidation were inhibited by DNP; the concentration of DNP required for inhibition of respiration was fortyfold higher than that required for inhibition of transport. Using the technique of equilibrium dialysis with membrane residues from which the lipid had been extracted, no binding of [¹⁴C]DMO to membrane protein was detected. However, [¹⁴C]DNP did bind to membrane protein. At 100 μm DNP, 12% of the [¹⁴C]DNP was bound, equivalent to 1.56 nmol/mg protein. The pH inside vesicles respiring on l-lactate was calculated from the distribution of [¹⁴C]DMO and was found not to differ from the pH of the suspending buffer. The mechanism of action of DNP on active transport in M. denitrificans vesicles appears not to involve proton conduction.     

Degradation of <Emphasis Type="Italic">p</Emphasis>-Toluenesulfonate by Immobilized <Emphasis Type="Italic">Comamonas testosteroni</Emphasis> BS1310 (pBS1010) Cells

Parameters of degradation of p-toluenesulfonate (TS) by free and agar-embedded Comamonas testosteroni BS1310 (pBS1010) cells were determined. The maximum rate of TS degradation was 25% lower in immobilized than free cells, equaling 11 nmol min^?1 mg^?1 cells. Degradation of TS by both free and immobilized cells was associated with molecular oxygen consumption (molar ratio 1 : 2). In a plug-flow reactor, the degradation rate was 10.4 nmol min^?1 mg^?1 cells. The results can be applied to designing reactors for TS degradation in sewage and developing biosensors.     

Transport of α-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens

The transport of α-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for α-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting α-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using ¹³⁷Cs in the presence of valinomycin. Direct attempts to demonstrate a proton-symport mechanism for α-aminoisobutyrate transport were negative.     

Amino Acid transport into membrane vesicles isolated from zucchini : evidence of a proton-amino Acid symport in the plasmalemma

Several lines of evidence with intact tissues suggest amino acid transport is mediated by a proton-amino acid symport (L Rheinhold, A Kaplan 1984 Annu Rev Plant Physiol 35: 45-83). However, biochemical studies of proton-coupled amino acid transport in isolated membrane vesicles have not been reported. In the experiments presented here, amino acid transport was studied in membrane vesicles isolated from zucchini (Cucurbita pepo L. cv Black Beauty) hypocotyls. An imposed pH gradient (basic interior) was used to energize isolated membrane vesicles and drive amino acid transport. Proton-coupled amino acid accumulation was demonstrated for alanine, glutamate, glutamine, leucine, and tabtoxinine-β-lactam. Alanine transport into the isolated membrane vesicles was studied in detail. Alanine transport was protonophore sensitive and accumulation ratios exceeding 10 times that predicted by diffusion alone were observed. ΔpH-Dependent alanine transport exhibited saturation kinetics, suggesting translocation was mediated via a carrier transport system. In support of that conclusion, 50 micromolar N,N′-dicyclohexylcarbodiimide, a hydrophobic modifier of protein carboxyls, completely inhibited proton-coupled alanine accumulation. Transport activity, equilibrated on a linear sucrose gradient, peaked at 1.16 grams per cubic centimeter and co-migrated with a plasmalemma marker (vanadate-sensitive K⁺-Mg²⁺-ATPase). These results provide direct evidence in support of a proton-amino acid symport in the plasmalemma of higher plants.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.     

Solubilization of a functionally active proline carrier from membranes of Escherichia coli with an organic solvent.

A proline transport carrier was extracted from the membranes of Escherichia coli with acidic n-butanol. Vesicles reconstituted from the butanol extract and E. coli phospholipids and preloaded with K⁺ showed rapid uphill uptake of proline when energy was supplied as a membrane potential introduced by K⁺-diffusion via valinomycin. Proline uptake by the reconstituted vesicles, like that of intact cells and isolated membrane vesicles, was inhibited by 3,4-dehydroproline, SH reagents, and a proton conducting uncoupler. Reconstituted vesicles of mutants defective in proline transport showed little or no proline uptake. The proline carrier was partially purified from the extract and separated from the bulk of phospholipids on Sephadex LH-20.     

Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H⁺-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO₃⁻ sensitive H⁺ transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H⁺ transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H⁺ transport capacity after adaptation was also observed for ATP hydrolysis.     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

Uptake of beta-ecdysone by the fat body and membrane vesicles of fat body cells of Sarcophaga peregrina larvae.

The fat body of Sarcophaga peregrina larvae was shown to incorporate ³H-β-ecdysone when it was incubated with the hormone in vitro. Most of the incorporated radioactivity was found in the cytoplasmic fraction as free β-ecdysone, not as a protein-β-ecdysone complex.Rapid uptake and accumulation of β-ecdysone was observed in the membrane vesicles of fat body cells in vitro. The apparent K_m value for uptake was estimated to be 1·25 × 10^?7 M. The β-ecdysone in the membrane vesicles was rapidly released when 2,4-dinitrophenol was added. These results suggest that β-ecdysone was incorporated into the membrane vesicles by active transport and not by free diffusion. The hormone is probably incorporated into larval tissues by the same mechanism as it is incorporated into the membrane vesicles of fat body cells.     

A novel method to quantify H-ATPase-dependent Na transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na⁺ is excluded from the cytosol to the apoplast or the vacuole by Na⁺/H⁺ antiporters. The secondary active transport of Na⁺ to apoplast against its electrochemical gradient is driven by plasma membrane H⁺-ATPases that hydrolyze ATP and pump H⁺ across the plasma membrane. Current methods to determine Na⁺ flux rely either on the use of Na-isotopes (²²Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H⁺ gradient due to H⁺-ATPase in the presence or absence of Na⁺ by pH-sensitive probes. To date, there are no methods that can directly quantify H⁺-ATPase-dependent Na⁺ transport in plasma membrane vesicles. We developed a method to measure bidirectional H⁺-ATPase-dependent Na⁺ transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H⁺-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na⁺ sealed for the study of H⁺-ATPase-dependent Na⁺ transport. Our data implicate that Na⁺ movement across vesicle membranes is highly dependent on H⁺-ATPase activity requiring ATP and Mg²⁺ and displays optimum rates of 2.50 μM Na⁺ mg^− 1 membrane protein min^− 1 at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H⁺-ATPase-dependent Na⁺ transport. The results demonstrate that the Na⁺ content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H⁺-ATPase-dependent Na⁺ transport with membrane vesicles.     

pH profiles of proline transport with membrane vesicles from Mycobacterium phlei with artificial and natural electron donors

Proline transport and rates of oxidation with artifical and natural electron donors were examined with membrane vesicles from $\text{M.}$$\text{phlei}$ as a function of pH. The levels of transport and rates of oxidation were parallel with generated NADH as substrate. With ascorbate-TPD, both the rate and level of transport increased from pH 7.5 to 9.0. The rate of total oxygen consumption with ascorbate-TPD correlated with the transport studies. However, when oxygen consumption was corrected for the auto-oxidation of ascorbate-TPD, the corrected oxygen consumption did not correlate with transport. Rates of cytochrome reduction were examined with ascorbate-TPD as electron donors to determine respiratory chain oxidation. For cytochromes c and a + a₃, the rates of reduction decreased as a function of pH, while active transport of proline increased.     

The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells

(1) The apparent K_m for nitrate of the electron-transport system in intact cells of Paracoccus denitrificans was less than 5 μM. In contrast the apparent K_m for nitrate of inverted membrane vesicles oxidising NADH was greater than 50 μM. When azide, a competitive inhibitor, was present, the apparent K_m observed with the vesicles was raised to 0.64 mM, consistent with values previously reported for purified preparations of the reductase. In membrane vesicles the nitrate reductase is probably not rate-limiting for NADH-nitrate oxido-reductase activity, and thus a lower limit for K_m (NO₃⁻) is obtained. It is suggested that the very low K_m (NO₃⁻) in intact cells must arise from either a transport process or a nitrate-specific pore that allows access of nitrate directly to the active site of its reductase from the periplasm. (2) The swelling of spheroplasts has been studied under both aerobic and anaerobic conditions to probe possible mechanisms of nitrate and nitrite transport across the plasma membrane of P. denitrificans. Nitrate reductase was inhibited by azide to prevent reduction of internal nitrate. No evidence for operation of either nitrate-nitrite antiport or proton-nitrate symport was obtained. (3) Measurements from the fluorescence intensity of 8-anilino-naphthalene-1-sulphonate of the rates of decay of diffusion potentials generated by addition of potassium salts to valinomycin-treated plasma membrane vesicles from P. denitrificans showed that the permeability of the membrane to anions is SCN⁻ > NO₃⁻, NO₂⁻, pyruvate, acetate > CI⁻ > SO₄²⁻. In the presence of a protonophore the rate of decay of the diffusion potential was considerably enhanced with potassium acetate or potassium nitrite, but not with potassium salts of nitrate, chloride or pyruvate. This result indicates that HNO₂ and CH₃COOH can rapidly and passively diffuse across the cell membrane. This finding suggests that transport systems for nitrite are in general probably not required in bacteria. The failure of a protonophore to enhance the dissipation of the diffusion potential generated by potassium nitrate is evidence against the operation of a proton-nitrate symporter. (4) Low concentrations of added nitrite very strongly inhibit electron flow to oxygen in anaerobically grown cells, provided that they have been treated with Triton X-100 or an uncoupler. This inhibition is not observed with aerobically grown cells. It is concluded that the inhibitory species is a reaction product or an intermediate of the nitrite reductase reaction. The requirement for collapse of protonomotive force by uncoupler or permeabilising the plasma membrane suggests that any such species could be negatively charged. Nitroxyl anion (NO⁻) can be considered, as its conjugate acid is a postulated intermediate between nitrite and nitrous oxide; nitroxyl anion can bind to heme centres to give nitrosyl derivatives. (5) The basis for the ability of permeabilised, but not intact, cells of P. denitrificans to reduce oxygen and nitrate simultaneously is discussed.     

Transport properties of bean vesicles in the presence of fungal components

Sealed membrane vesicles were isolated from hypocotyls of two varieties of French bean (Phaseolus vulgaris). The preparations were shown to contain vesicles in which H⁺ transport and ATPase were sensitive to nitrate but insensitive to vanadate. These sensitivities suggest the vesicles were enriched for tonoplast. Fractions prepared from the α and β races of the fungus Colletotrichum lindemuthianum inhibited H⁺ transport in the vesicles isolated from cvs. Dark Red Kidney and Great Northern bean tissue. These data are discussed in terms of the biochemical mechanisms operating in plant/pathogen interactions.     

The generation of singlet oxygen (o(2)) by the nitrodiphenyl ether herbicide oxyfluorfen is independent of photosynthesis

The mechanism of action of the p-nitrodiphenyl ether herbicides has remained ambiguous because of conflicting reports in the literature. The diphenyl ether herbicide oxyfluorfen causes a light induced consumption of oxygen which resembles the electron acceptor reaction of paraquat. However, this reaction is not linked to the transport of electrons through photosystem I. This conclusion is based on the observation that the rate of oxygen consumption, in the presence of oxyfluorfen, does not demonstrate a first order rate dependence on light intensity. Using the bleaching of N,N-dimethyl p-nitrosoaniline as a specific detector of singlet oxygen, we demonstrate that oxyfluorfen is a potent generator of this toxic radical. The production of singlet oxygen occurs in the presence of inhibitors of photosynthetic electron transport (oxyfluorfen at 10⁻⁴ molar and paraquat) and also under temperature conditions (3°C) which prevent electron transport. This light induced reaction results in oxygen consumption and is the primary cause of lethality for oxyfluorfen. The production of singlet oxygen occurs rapidly and at low herbicide concentrations (10⁻⁹ molar). The reaction occurs without photosynthetic electron transport but does require an intact thylakoid membrane.     

Glutamate,aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain

To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K_m of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K⁺ and Rb⁺ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na⁺; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na⁺ and K⁺ gradients. Monensin inhibits uptake by selectively dissipating the Na⁺ gradient. For both glutamate and GABA transport, the Na⁺ and K⁺ gradients are synergistic and not additive.