Inside Alkaline Research Articles

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na + gradient across the membrane was imposed. The maximum effect of Na + on the transport was achieved at a concentration of about 40 mM, while the apparent K m for Na + was approximately 8 mM. On the other hand, K m for glutamate in the presence of 50 mM Na + was about 8 μM. Increasing the concentration of Na + resulted in a decrease in K m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na + ionophore). Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K +-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K m for glutamate was approximately 25 μM. These results indicate that two kinds of glutamate transport system were present in H protein: one is Na + dependent and the other is H + dependent.

Relationship between proton motive force and potassium Ion transport in Halobacterium halobium envelope vesicles

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na + extrusion through an antiport system, and the ability of K + and Cl − to act as counterions to the electrogenic movement of H +. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K +. The vesicle membrane is poorly permeable to K +, as shown by: lack of a K + diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K +, and direct measurements of the slow rate of K + influx during illumination. The finding that the rate of K + uptake is a linear function of external K + concentration between 0 and 1 m is inconsistent with the existence of a specific K + permeation mechanism in these vesicles. Since at external K + concentrations < 1.4 m the extrusion of Na + during illumination proceeds much more rapidly than K + influx, it must be concluded that the vesicles also lose Cl − and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K + is increased, either by increasing the external K + concentration or by adding valinomycin.

Resolution of microsomal membrane proteins by two-dimensional gel electrophoresis [proceedings

the citrate medium described by Halestrap (1976) was used, since this generated a pH gradient (alkaline inside) which aided pyruvate and lactate accumulation. In other experiments buffered saline was used as described by Spencer & Lehninger (1976) in order to resemble the physiological situation more closely. Details of the conditions are given in the legends to the Figure and Table. In Fig. 1 the inhibition of pyruvate transport by mersalyl and p-chloromercuribenzenesulphonate is demonstrated; similar results were obtained when L-lactate was the substrate for transport. Both reagents are potent inhibitors of pyruvate transport, p-chloromercuribenzenesulphonate being the more powerful. The inhibition by mersalyl appears to be similar to that observed by Spencer & Lehninger (1976) for ascites cells, although the inhibitor is more potent in the erythrocyte. This may reflect the higher pH (7.4) used in the present studies. The inhibition by p-chloromercuribenzenesulphonate is similar to that observed by Deuticke et al. (1978) in human erythrocytes. Table 1 shows that under similar conditions to those used by Halestrap (1976), similar kinetic constants were obtained. Thus the presence of 4-acetamide-4’-isothiocyanatostilbene-2,2’-disulphonate, which inhibited C1--dependent pyruvate or lactate uptake, altered the kinetics very little at low substrate concentrations where transport on the Clcarrier is slow. In saline buffer the K,,, values were similar to those in citrate buffer, but the VmaX. values were considerably lower when temperature differences were taken into account. Under these conditions, kinetic parameters were the same whether measured by ‘inhibitor stop’ or proton-flux techniques, indicating that net carboxylate uptake occurs with a proton. Outdated blood studied in citrate buffer shows no proton fluxes and carboxylate uptake occurs in exchange for internal lactate (Halestrap, 1976). Since exchange is usually faster than net flux (see Halestrap, 1978), this may explain the difference in V,,,,,, values. Activation energies measured under both conditions are similar, but the Ki for a-cyano-4-hydroxycinnamate is much higher in saline buffer than in citrate buffer. This may be due to the existence of a pH gradient (alkaline inside) in cells incubated in citrate buffer (Halestrap, 1976) which may increase inhibition by a-cyano-4-hydroxycinnamate through concentration of the inhibitor inside the cell. The data presented for experiments in saline buffer suggest that the C1--independent carrier for pyruvate and lactate in the erythrocyte is similar to that described by Spencer & Lehninger (1976) for ascites cells.

The electrochemical proton gradient generated by light in membrane vesicles and chromatophores from Rhodopseudomonas sphaeroides.

The electrochemical proton gradient, , generated upon illumination of membrane vesicles and chromatophores from Rhodopseudomonas sphaeroides. has been determined. The components, the transmembrane pH gradient, ΔpH, and the membrane potential, ΔΨ, were measured with several methods. The ΔpH, determined from the pH changes in the membrane suspensions, is in good agreement with the ΔpH obtained from the distribution of acetate or methylamine. The ΔpH values obtained from the fluorescence quenching of 9‐aminoacridine in chromatophores are more than two‐fold larger. The ΔΨ was determined from the distribution of the permeant ions triphenylmethylphosphonium and thiocyanate. The ΔΨ values in chromatophores obtained from the absorbance changes of carotenoids were several‐fold larger.Illumination of membrane vesicles results in a ΔpH, inside alkaline and a ΔΨ inside negative. The ΔpH, determined from the distribution of acetate, depends on the composition of the medium and is optimally 0.6 at an external pH 7; the ΔΨ, measured from the distribution of triphenyl‐methylphosphonium, is about 70 mV. The in membrane vesicles is thus at the most 110 mV.Illumination of chromatophores results in a ΔpH, inside acid, and a ΔΨ inside positive. The ΔpH, calculated from the distribution of methylamine, depends on the external pH and increases from 0.98 at pH 6 to 1.36 at pH 8. The ΔΨ is not affected and remains about 50 mV. The in chromatophores is thus at the most 140 mV.The ΔpH and ΔΨ are in both membrane preparations strongly dependent on the composition of the medium. So is for instance the ΔΨ decreased by the presence of chloride ions and the ΔpH increased. Phosphate ions decrease the ΔpH in membrane vesicles. The ΔpH is generated at a lower rate than the ΔΨ; once the has reached a maximum the ΔpH continues to increase at the expense of the ΔΨ.

The protonmotive force and alpha-aminoisobutyric acid transport in an obligately alkalophilic bacterium.

Measurements of the protonmotive force and studies of solute transport in Bacillus alcalophilus were conducted to ascertain how ceil processes are energized under conditions in which a pH gradient, interior alkaline, cannot be established across the membrane. B. alcalophilus grows on Lmaiate in a range of pH from 9.0 to 11.5, with an optimum at pH 10.5. The electrical potential across the membrane, A+, increases progressively from -84 mV at pH 9.0 to -152 mV at pH 11.5. At pH 10.0 and above a ApH exists, interior acid, increasing from 36 mV at pH 10.0 to 151 mV at pH 11.5. At pH 10.5 and 11, at which growth is excellent, the total transmembrane protonmotive forces were only -80 mV and -15 mV, respectively. Uptake of cu-aminoisobutyric acid (AIB), to concentrations 845-fold greater than the external concentration, occurs only at alkaline pH values, with an optimum at pH 10.5. Saturation kinetics, with a K, for AIB of 9.3 JAM, were

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.

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg 2+ dependent; Co 2+ and Mn 2+ but not Ca 2+ could replace Mg 2+ to some extent; the activation by Mg 2+ was slightly antagonized by Ca 2+. Even in the presence of Mg 2+, Na + or K + had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S] 0.5 V = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg 2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10 -4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN 3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H + translocation. A H +-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H +-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.

Protonmotive force as the source of energy for adenosine 5'-triphosphate synthesis in Escherichia coli.

Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974).

ATP synthesis driven by a protonmotive force inStreptococcus lactis

An electrochemical potential difference for hydrogen ions ( a protonmotive force) was artifically imposed across the membrane of the anaerobic bacterium Streptococcus lactis. When cells were exposed to the ionophore, valinomycin, the electrical gradient was established by a potassium diffusion potential. A chemical gradient of protons was established by manipulating the transmembrane pH gradient. When the protonmotive force attained a value of 215 mV or greater, net ATP synthesis was catalyzed by the membrane-bound Ca++, Mg++ -stimulated ATPase. This was true whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Under these conditions, ATP synthesis could be blocked by the ATPase inhibitor, dicyclohexylcarbodiimide, or by ionophores which rendered the membrane specifically permeable to protons. These observations provide strong evidence in support of the chemiosmotic hypothesis, which states that the membrane-bound ATPase couples the inward movement of protons to the synthesis of ATP.

Phosphate transport in membrane vesicles of Paracoccus denitrificans

The mitochondrial Pi carrier mediates the electroneutral, reversible exchange of Pi for OHacross the inner mitochondrial membrane [ 1,2] . Thus Pi uptake into the mitochondrion occurs primarily in response to the pH gradient (alkaline inside) imposed across the mitochondrial membrane [3] ; this is effectively the proton symport of Mitchell [4]. In bacteria the mechanism of Pi uptake is less well understood. In Staphylococcus aureus and escherichia coli [4] and in Streptococcus faecalis [5] it has been proposed that Pi uptake occurs by an electroneutral exchange of Pi for OHacross the bacterial plasma membrane, but in none of the bacteria so far investigated has Pi uptake in response to a pH gradient been demonstrated. The plasma membrane of Paracoccus denitrificans, previously Micrococcus denitrificans [6] , is already known to possess many features of the mitochondrial inner membrane [7], and this prompted us to determine whether P. denitrificans also possesses the same type of Pi carrier as the mitochondrion. In the present paper we show that Pi uptake into membrane vesicles prepared from the plasma membrane of P. denitrificans can be driven by a pH gradient (alkaline inside) applied across the vesicle membrane. We also show that the Pi carrier present in these vesicles (again like the mitochondrial Pi carrier) is reversible and sensitive to SH-group reagents.

The reversibility of active sulphate transport in membrane vesicles of Paracoccus denitrificans.

An uncoupler-sensitive active transport of sulphate into membrane vesicles prepared from the plasma membrane of Paracoccus denitrificans (previously Micrococcus denitrificans) can be driven by respiration or by a trans-membrane pH gradient (alkaline inside) generated by the addition either of KCL ( in the presence of nigericin) or of NH4CL. Valinomycin does not substitute for nigericin. Respiration-driven transport is observed in right-side-out vesicles but not in inside-out vesicles, whereas transport driven by the addition of KCL (in the presence of nigericin) or of NH4CL is observed in both types of membrane vesicle. The active transport of sulphate into these vesicles is shown to be carrier-mediated by its sensitivity to thiol-group reagents. It is proposed that the sulphate carrier in the plasma membrane of P. denitrificans operates by a mechanism of electroneutral proton symport, and is capable of actively transporting sulphate in either direction across the plasma membrane, but that in whole cells respiration-driven proton expulsion drives the accumulative uptake of sulphate.

The internal-alkaline pH gradient, sensitive to uncoupler and ATPase inhibitor, in growing Clostridium pasteurianum.

1. The intracellular pH was measured in growing Clostridium pasteurianum with and acid-base equilibrium distribution method. [14C]Dimethyloxazolidinedione, [14]methylamine and [14C]acetic acid were used as "deltapH-indicators". During growth the extracellular pH decreased from 7.1 to 5.1; simultaneously the intracellular pH changed from 7.5 to 5.9. Thus, the intracellular pH was more alkaline than the extracellular pH by 0.4 to 0.8 pH-units. 2. This pH gradient (interior alkaline) was abolished by the proton conductor carbonylcyanide m-chlorophenylhydrazone and the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The pH gradient could not be demonstrated in cells depleted of an energy substrate. These results suggest that the pH gradient is formed by an ATPase-driven extrusion of protons from the cells rather than by a Donnan potential. 3. Growth of the organism was inhibited by low concentrations of both carbonylcyanide m-chlorophenylhydrazone (5 muM) and dicyclohexylcarbodiimide (5 muM). This finding suggests that the pH gradient is essential for the growing cell as it may be required for substrate accumulation and other types of transport processes.

Mechanisms of energy coupling to the transport of amino acids by Staphylococcus aureus.

We have studied the effect of artifically imposed transmembrane pH gradients on the uptake of amino acids by Staphylococcus aureus. In high potassium medium, cells treated with valinomycin and tetrachlorosalicylanilide and at Donnan equilibrium, were found to accumulate glutamate. In low potassium medium, addition of acid to the extracellular environment was found to stimulate the uptake of glutamate and aspartate, to induce partial lysine efflux, and to cause a small and transient isoleucine uptake. In conjunction with earlier data from this laboratory (1973), it is concluded that the basic, neutral and acidic amino acids behave, respectively, as cationic, uncharged and anionic sub-strates, accumulating in Staphylococcus aureus in response to (a) the membrane potential, inside negative, (b) the total protonmotive force, and (c) the transmembrane pH gradient, inside alkaline.

The membrane potential as the driving force for the accumulation of lysine by Staphylococcus aureus

One of the most significant attributes of the chemiosmotic hypothesis [l] is that it relates directly not only to oxidative phosphorylation, but to all of the many energetic and control functions associated with cellular membranes. This feature is particularly relevant in the study of energy coupling mechanisms in bacteria with their single membranous structure. In the field of transport, one would predict that both the major components of the protonmotive force, membrane potential and pH gradient, might be capable of driving the transport of ions and nutrients across membranes against their prevailing electrochemical gradients. A flux of cations might therefore be driven by the membrane potential (inside negative), while anion transport might respond to the pH gradient (inside alkaline) through the action of a proton-symport; such a cotransport with protons would give a positively charged entity with, for example, the neutral sugars, which could then respond to both the membrane potential and the pH gradient [2,3]. We have set out to test this prediction with respect to the transport of the basic, neutral and acidic amino acids into the intracellular pool of Staphylococcus aureus. This paper deals specifically with our findings with the most experimentally accessible case, that of lysine, which, at neutral pH, exists predominantly as the positively charged species. Metabolically depleted cells, possessing relatively high proton and potassium permeabilities, maintain protons near Donnan equilibrium. In such cells there is a membrane potential which is largely an ionic diffusion potential; this may be varied by ionic manipulation, for example, by varying the extracellular potassium concentration in the presence of valinomycin. We have