Reduced Phenazine Methosulfate Research Articles

Fluorometric biosensing of α-amylase using an artificial allosteric biosensor immobilized on nanostructured interface

Protein biosensors hold a promise to transform the way we collect physiological data by enabling quantification of biomarkers outside of specialized laboratory environment. However, achieving high specificity and sensitivity in homogeneous assay format remains challenging. Here we report construction of fluorescent biosensor arrays based on artificial allosteric α-amylase-activated PQQ-dependent glucose dehydrogenase (Amy-GDH). Amy-GDH was covalently immobilized on silica nanoparticles that were then arrayed on fiberglass sheets. The activity of the biosensor was monitored using a smartphone camera via emergence of bright fluorescence (λex 365 nm) originating from reduced phenazine methosulfate upon glucose oxidation by Amy-GDH. We show that such biosensor arrays demonstrate an apparent Kd of 115 pM for α-amylase with a detection limit of 2 pM. Using the developed biosensor arrays, we were able to specifically and accurately quantify the concentration of α-amylase in biological fluids such as serum and saliva. We propose that the presented approach can enable construction of ultrasensitive point-of-care diagnostic arrays.

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate

Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.

L-Galactono-1,4-lactone dehydrogenase (GLDH) Forms Part of Three Subcomplexes of Mitochondrial Complex I in Arabidopsis thaliana

L-galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.

A Case of IgG kappa-LD Complex, Producing Long-Term Increase of Enzyme Activity and a Rare Isoenzyme Pattern

Background: Serum LD activity is increased in several diseases. The clinical significance of elevated LD may be evaluated by isoenzyme analysis. Here, we report on a long-term elevation of serum LD activity with a rare isoenzyme pattern caused by LD-IgG complexes. Patient, methods: A 75-year old female with atherosclerosis and implantations of two stents showed increased LD activity and an unusual isoenzyme pattern. LD activity, inhibition and immunoglobulins were measured with Roche tests; immunofixation was performed with a Helena test. LD isoenzymes were separated in agarose gel and quantified by the color formed by NADH, reduced phenazine methosulfate and nitro blue tetrazolium. Results: LD isoenzyme analyses revealed one band near the slowly migrating LD-4 position. The band contained LD complexed with immunoglobulins (IgG with κ light chains), The long-term increased serum LD enzyme activity in this patient may have been due to abnormally slow clearance of these large macro-LD complexes from circula...

Reactive oxygen species involved in phenazine-methosulfate-induced rat lens opacification. An experimental model of cataract.

The excised rat crystalline lens opacified when incubated aerobically with phenazine methosulfate, but no opacification was observed under anaerobic conditions. Morphological studies revealed development of opacification in the cortex. The opacification resembled that often seen in the early period of senile cataract as well as in naphthalene-induced and UV cataract. Both an increase in hydration and in electrolyte imbalance accompanied this opacification. Na,K-ATPase activity of the opacified lens was found to decrease. In order to investigate if activated oxygen is involved in these processes, we conducted an electron spin resonance study by means of a spin trapping technique. When the lens homogate was incubated with phenazine methosulfate, OH radicals were generated under aerobic but not under anaerobic conditions. Reduced pyridine nucleotides must be involved in the process, because the mixture of nicotinamide adenine dinucleotide phosphate [NAD(P)] and phenazine methosulfate did not generate OH radicals, but the mixture of NAD(P)H and phenazine methosulfate generates OH radicals, indicating that reduced phenazine methosulfate was involved in the OH radical generation. Probably, the generated OH radicals inactivated Na,K-ATPase residing in the epithelium of the lens, which eventually caused opacification of the lens. The present experiment system may be used for the elucidation of lens opacification (cataract) involved with reactive oxygen species.

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.

Enhancement of superoxide production in vitro by the diphenyl ether herbicide nitrofen

Light-dependent reduction of p-nitro blue tetrazolium chloride (NBT) and 2,6-dichlorophenol-indophenol (DCPIP) was stimulated by nitrofen (2,4-dichlorophenyl p-nitrophenyl ether) in the presence or absence of the photosensitizer riboflavin. Enhancement of NBT reduction occurred at a concentration as low as 17 μ M nitrofen. Nitrofen had no effect on dark reduction of NBT by dithionite, ascorbate, or reduced phenazine methosulfate. NBT reduction proceeded in solution saturated with either air or nitrogen. Stimulation by nitrofen in the presence or absence of riboflavin also occurred independently of oxygen. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1) inhibited light-dependent, aerobic reduction of NBT and DCPIP with and without riboflavin. The nitrogen-stimulated component was also eliminated. Thus, it appears nitrofen can effect reduction of NBT (and DCPIP) anaerobically by transferring electrons directly to NBT (and DCPIP) through an oxygen-independent mechanism or aerobically via superoxide radicals.

Reconstitution of electrogenic function in isolated pigment-protein complexes of Anabaena variabilis

Treatment of Anabaena variabilis membranes with lauryldimethylamine N-oxide yielded two fractions of pigment-protein complexes which were separable by gel filtration on Sepharose 6B. A green fraction was characterized which had a maximum of the chlorophyll long-wave absorption band at 678 nm and a small amount of carotenoid. In this fraction, Photosystem I activity was higher than in another (brownish-green) fraction which had a maximum of the chlorophyll absorption band at 673 nm and which was enriched in carotenoids. Similarly to isolated membranes, proteoliposomes containing pigment-protein complexes took up tetraphenylborate anions and tetraphenylphosphonium cations and were found to be capable of light-dependent membrane potential generation, when associated with a planar phospholipid membrane in the presence of reduced phenazine methosulfate upon illumination. The spatial arrangement of the pigment-protein complexes in the native and artificial membranes is discussed.

Active uptake of tetracycline by membrane vesicles from susceptible Escherichia coli.

A major portion of tetracycline accumulation by susceptible bacterial cells is energy dependent. Inner membrane vesicles prepared from susceptible Escherichia coli cells concentrated tetracycline 2.5 to 5 times above the external concentration when the electron transport substrate D-lactate or reduced phenazine methosulfate was added. This stimulation was reversed by cyanide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenyl hydrazone. These vesicles data showed that proton motive force alone could energize tetracycline uptake. The lactate-dependent uptake had a pH optimum of 6.9 and a magnesium optimum of 1 mM and was not saturable up to 400 microM tetracycline. Although the vesicles were not as active as cells in concentrating tetracycline, they were less active to a similar extent in concentrating tetracycline, they were less active to a similar extent in concentrating proline, the transport of which is known to be solely proton motive force dependent. Therefore, we concluded that the active uptake of tetracycline in susceptible cells was largely, if not solely, energized by proton motive force.

Effect of the proton electrochemical gradient on maleimide inactivation of active transport in Escherichia coli membrane vesicles.

Many active transport systems present in Escherichia coli membrane vesicles are inhibited by maleimides and other sulfhydryl reagents. These reagents do not interfere with the oxidation of reduced phenazine methosulfate or with the electrochemical proton gradient (delta muH+). The rate of inactivation is increased in the presence of reduced phenazine methosulfate, and it is shown that the electrochemical proton gradient is responsible for the effect. Furthermore, similar effects observed with the proline and melibiose transport systems. Thus, it appears that either the reactivity or accessibility of a sulfhydryl group(s) in each of these carriers is altered by the presence of a transmembrane delta muH+. The findings are consistent with the notion that delta muH+, in addition to acting as the immediate driving force the active transport, may bring about structural or conformational changes in certain membrane proteins that catalyze active transport.

Fluorescence and Photochemical Properties of Plants with Defective Photosystem II

Abstract The mykotrophic orchid Neottia nidus-avis does not evolve oxygen in the light but is able to perform photophosphorylation. The low temperature fluorescence emission spectrum lacks the 680 and 690 nm bands. Hence, the spectroscopic chlorophyll a forms which are attributed to photosystem II do not occur in plastids of this orchid. The low temperature excitation spectrum of photosystem I fluorescence exhibits a maximum at 666 nm. The position of this maximum appears not to be influenced by energy transfer and corresponds to the absorption maximum of the chlorophyll form which emits the photosystem I fluorescence. Energy migration, however, occurs from carotenoids whose absorption spectrum is shifted to longer wavelengths and which cause the yellow-brown color of the Neottia plastids. Room temperature fluorescence emission shows after the onset of light no variable part. Despite the fact that plastids of the tobacco mutant NC 95 at most evolve only traces of oxygen the low temperature emission spectrum shows the three bands which are usually observed with fully functioning chloroplasts. However, the two bands at 680 and 690 nm are distinctly lower than with the wild type. The variable portion of room temperature fluorescence is barely detectable. In line with the very low capacity for oxygen evolution, rates of electron transport partial reactions in the region of photosystem II are extremely low. In agreement with this observation no 690 nm absorption change signal is detected. However, a normal P+700 signal is seen. In the presence of electron donors like reduced phenazine methosulfate the decay time of the P+700 signal is faster than with the wild type. The yellow tobacco mutant Su/su var. aurea which exhibits at high light intensities higher rates of photosynthesis than the wild type shows at low temperature an emission spectrum with stronger photosystem II bands than the wild type.

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.

Electrochemical proton gradient in inverted membrane vesicles from Escherichia coli.

Inverted membrane vesicles prepared from Escherichia coli ML 308-225 generate a transmembrane electrochemical proton gradient (delta mu H+; interior positive and acid) during oxidation of D-lactate, succinate, reduced phenazine methosulfate, or NADH or hydrolysis of ATP. Using the distribution of the lipophilic anion thiocyanate to measure the membrane potential (delta psi) and the permeant weak base methylamine to measure the pH gradient (delta pH), maximal values for delta psi H+ of approximately +160 mV are obtained. Many of the properties of delta psi H+ in inverted vesicles are similar to those described previously in right-side-out vesicles [Ramos, S., & Kaback, H.R. (1977) Biochemistry 16, 848]: (1) the magnitude of the delta psi (interior positive) generated in the presence of D-lactate or reduced phenazine methosulfate is similar to that observed in right-side-out vesicles but of opposite polarity and independent of pH from 5.5 to 8.0; (2) plots of delta pH vs. internal pH in the right-side-out vesicles are similar with D-lactate as the electron donor; (3) as observed with right-side-out vesicles, dissipation of delta psi or delta pH leads to a concomitant increase in the other parameter without a change in the rate of respiration; (4) inverted vesicles catalyze Na+ accumulation, and it is apparent that the process can be driven by either delta psi (interior positive) or delta pH (interior acid).

Electron acceptors associated with P-700 in Triton solubilized Photosystem I particles from spinach chloroplasts

Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700 + with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 μs back reaction of P-700 +, which we associate with a more primary electron acceptor A 2. In turn, when A 2 is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 μs) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A 1, which functions very close to P-700. The organization of these components probably corresponds to the sequence P-700- A 1- A 2-P-430[ A B ] . The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed. Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.

Respiration-coupled calcium transport by membrane vesicles from Azotobacter vinelandii.

Membrane vesicles, isolated from osmotic lysates of Azotobacter vinelandii spheroplasts in Tris-acetate buffer, rapidly accumulate calcium in the presence of an oxidizable substrate. The addition of D-lactate to vesicles increases the rate of calcium uptake by 34-fold; L-malate, NADH, NADPH, and reduced phenazine methosulfate are nearly as effective as lactate. The intravesicular calcium pool which accumulates under these conditions is rapidly discharged by isotopic exchange or in the presence of respiratory inhibitors, uncouplers, or EGTA. The uptake rates for calcium follow Michaelis-Menten kinetics yielding a Km of 48 microM and a V max of 45 nmoles/min/mg membrane protein. Initial rates of EGTA-induced calcium efflux also follow saturation kinetics, giving a V max identical to that for calcium entry; but the Km for exodus is 14 mM, assuming that free calcium accumulates in vesicles. The difference in the affinity of calcium for the entry and exit processes observed during respiration is sufficient to account for the estimated 150-fold calcium concentration gradient achieved under steady-state conditions. The uptake system is specific for calcium as opposed to other cations, but zinc and lanthanum are effective competitors. Calcium uptake is blocked when electron is inhibited by exposure of vesicles to p-chlormercuriphenylsulfonate, hydroxyquinoline-N-oxide, or cyanide, or under anoxic conditions. Divalent cation ionophores (A23187 and X537A) and proton ionophores (CCP and gramicidin D) also block calcium transport effectively. The electrogenic potassium ionophore valinomycin has no effect on lactate-dependent calcium uptake in the presence of potassium; but ionophores which induce electroneutral exchange of protons for sodium or potassium (monensin and nigericin, respectively) did block calcium transport in the presence of the appropriate cation. The fluorescence intensity of quinacrine (an amine probe) in the presence of A. vinelandii membrane vesicles is reduced by 25% on addition of lactate; the quenching is blocked by CCP. This indicates that a pH gradient (inside acid) is developed across the vesicle membrane during lactate oxidation. These results indicate that these membrane preparations contain vesicles of inverted topology (with respect to the intact cell) and suggest that calcium transport occurs by means of electroneutral calcium/proton antiport.

The electrochemical proton gradient in Escherichia coli membrane vesicles.

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.

Electron transport between plastoquinone and chlorophyll A1 in chloroplasts. II. Reaction kinetics and the function of plastocyanin in situ

The light-induced reaction kinetics of electron carriers between the two light reactions were studied in spinach chloroplasts. 1. 1. The difference spectrum of the absorbance changes of plastocyanin in situ was separated from superimposing absorbance changes by flash titration described in the preceding paper (Haehnel, W. (1973) Biochim. Biophys. Acta 305, 618–631). Relative amounts of 2:1 electron equivalents were observed for plastocyanin and chlorophyll a I ( P-700). 2. 2. A balance of the electron equivalents released from reduced plastoquinone and simultaneously accepted by oxidized plastocyanin, cytochrome f and chlorophyll a I indicated a quantitative electron transfer. Additional electron carriers between plastoquinone and light reaction I can be excluded with an accuracy of about ±0.3 electron equivalents per light reaction II. 3. 3. The time course of the absorbance changes of plastocyanin was measured at 584 nm with negligible interference with other absorbance changes. The reduction kinetics show an initial lag followed by a rise with a half time of about 20 ms. The redox states of plastocyanin and chlorophyll a I during this reduction via the rate-limiting step between the light reactions and during oxidation by weak far-red light suggest a true equilibrium constant of about 20. 4. 4. The simultaneous oxidation and reduction kinetics of plastoquinone, cytochrome f, plastocyanin and chlorophyll a I induced by two successive groups of saturating flashes after far-red illumination were measured. The oxidation kinetics of plastocyanin and the simultaneous reduction kinetics of chlorophyll a I after the single flashes indicate a quantitative electron transfer with a half time of 200 μs. 5. 5. The fast reduction of chlorophyll a I by plastocyanin showed no effect of the inhibitors 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone or of reduced phenazine methosulfate. But it was completely inhibited after KCN incubation. 6. 6. The oxidation kinetics of cytochrome f were reinvestigated with high time resolution from the difference of absorbance changes at 554 minus 540 nm to minimize the disturbing interference with other absorbance changes. Absorbance changes measured at 554 nm alone do not reflect kinetics of cytochrome f. The half time of the oxidation was faster than 40 μs. 7. 7. The observed reaction kinetics gave evidence for a function of cytochrome f between plastoquinone and chlorophyll a I in parallel to plastocyanin. In addition, they indicate that the greater portion of linear electron transport passes plastocyanin. The complex interaction between cytochrome f and chlorophyll a I is discussed.

Functional mosaicism of membrane proteins in vesicles of Escherichia coli

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.

Orientation of the protonmotive force in membrane vesicles of Escherichia coli.

AbstractMembrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D‐lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside.The latter type of vesicle, on the other hand, exhibits D‐lactate‐dependent proline transport but does not accumulate calcium with D‐lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D‐lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy‐linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).

Energetics and molecular biology of active transport in bacterial membrane vesicles

Bacterial membrane vesicles retain the same sidedness as the membrane in the intact cell and catalyze active transport of many solutes by a respiration-dependent mechanism that does not involve the generation of utilization of ATP or other high-energy phosphate compounds. In E. coli vesicles, most of these transport systems are coupled to an electrochemical gradient of protons (deltamuH+, interior negative and alkaline) generated primarily by the oxidation of D-lactate or reduced phenazine methosulfate via a membrane-bound respiratory chain. Oxygen or, under appropriate conditions, fumarate or nitrate can function as terminal electron acceptors, and the site at which deltamuH+ is generated is located before cytochrome b1 in the respiratory chain. Certain (N-dansyl)aminoalkyl-beta-D-galactopyranosides (Dns-gal) and N(2-nitro-4-azidophenyl)aminoalkyl 1-thio-beta-D-galactopyranosides (APG) are competitive inhibitors of lactose transport but are not transported themselves. Various fluorescence techniques, direct binding assays, and photoinactivation studies demonstrate that the great bulk of the lac carrier protein (ca. 95%) does not bind ligand in the absence of energy-coupling. Upon generation of a deltamuH+ (interior negative and alkaline), binding of Dns-gal and APG-dependent photoinactivation are observed. The data indicate that energy is coupled to the initial step in the transport process, and suggest that the lac carrier protein may be negatively charged.