Preparation of reconstituted cytochrome oxidase vesicles with defined trans-membrane protein orientations employing a cytochrome c affinity column

Reconstituted cytochrome oxidase systems in which the majority of the vesicles contain a single oxidase dimer can be prepared. It is shown that, when these are passed through a cytochrome c affinity column, only those vesicles oriented outwards (such that the active site is available to external cytochrome c) are bound to the support matrix. Protein-free vesicles and vesicles containing an inwardly oriented enzyme are eluted in the void volume. Subsequently, vesicles containing an outwardly oriented enzyme can be eluted from the column at high salt concentrations. This protocol has been used successfully to resolve vesicles of either oxidase orientation when the enzyme is reconstituted with a variety of lipid mixtures. The recovery of oxidase activity from the column ranged between 75 and 94%.     

Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase

Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.     

Effect of gangliosides on membrane permeability studied by enzymic and fluorescence-spectroscopy techniques.

The effect of gangliosides on membrane permeability was investigated by studying the kinetic properties of cytochrome c oxidase, the activity of which, when the enzyme is reconstituted in phospholipid vesicles, is dependent on membrane permeability to H+ and K+. The experiments indicate that three different gangliosides (GM1, DD1a, GT1b) incorporated into cytochrome c oxidase-containing phospholipid vesicles stimulate enzymic activity, in the absence of ionophores, most probably by disorganizing the bilayer lipid assembly and increasing its permeability to ions. This interpretation was confirmed by fluorescence-spectroscopy experiments in which the rate of passive leakage of carboxyfluorescein entrapped in the vesicles was measured. Cholera toxin, or its isolated B-subunit, added to GM1-containing proteoliposomes inhibited cytochrome c oxidase activity, indicating the lack of formation, under these experimental conditions, of channels freely permeable to H+ or K+.     

Influence of detergent polar and apolar structure upon the temperature dependence of beef heart cytochrome c oxidase activity

The temperature dependence of lipid-depleted beef heart cytochrome c oxidase activity was studied in a series of chemically homogeneous detergents. The detergents that were tested included C10 to C18 maltosides, C8 to C12 glucosides, C8 to C16 Zwittergents, and C12 poly(oxyethylene) ethers. The observed rates of electron transport were dependent upon the structure of the polar head group and the length of the hydrocarbon tail. Of the detergents tested, the alkyl maltosides were the best in terms of both high rates of electron transport and superior enzyme stability. With the maltosides, changing the length of the alkyl tail affected the activity of cytochrome c oxidase in a manner quite similar to that reported with synthetic phosphatidylcholines and phosphatidylethanolamines [Vik, S. B., & Capaldi, R. A. (1977) Biochemistry 16, 5755-5759], suggesting that the alkyl maltosides can mimic some of the features of the membrane environment. In each of the detergents, the activation enthalpy (determined from the slope of an Arrhenius plot) was nearly identical, suggesting that the same electron-transfer step within cytochrome c oxidase is rate limiting. This result has been interpreted as evidence for the existence of two or more conformers of cytochrome c oxidase, one of which is significantly more active than the other(s). The enzyme turnover number, which changes by 2 orders of magnitude depending upon the structure of the bound detergent, may reflect the ability of each detergent to alter the equilibrium between the active and nearly inactive conformers.     

The subcellular distribution of adenylate and guanylate cyclases in murine lymphoid cells.

Membrane vesicles can be prepared from murine lymphoid cells by nitrogen cavitation and fractionated by sedimentation through nonlinear sucrose density gradients. Two subpopulations of membrane vesicles, PMI and PMII, can be distinguished on the basis of sedimentation rate. The subcellular distribution of adenylate and guanylate cyclases in these membrane subpopulations have been compared with the distribution of a number of marker enzymes. Approximately 20-30% of the total adenylate and guanylate cyclase activity is located at the top of the sucrose gradient (soluble enzyme), the remainder of the activity being distributed in the PMI and PMII fractions (membrane-bound enzyme). More than 90% of the 5'-nucleotidase and NADH oxidase activities detected in lymphoid cell homogenates are located in PMI and PMII fractions, whereas succinate cytochrome c reductase activity is detected only in the PMII fractions. In addition, beta-galactosidase activity is distributed in the soluble and PMII fractions of the sucrose density gradients. On the basis of the fractionation patterns of these various enzyme activities, it appears that PMI fractions contain vesicles of plasma membrane and endoplasmic reticulum, whereas PMII fractions contain mitochondria, lysomes, and plasma membrane vesicles. Approximately 30-40% of the adenylate and guanylate cyclase activities in PMII can be converted to a PMI-like form following dialysis and resedimentation through a second nonlinear sucrose gradient. Adenylate and guanulate cyclases can be distinguished on the basis of sensitivity to nonionic detergents.     

Ionic-strength-dependence of the oxidation of native and pyridoxal 5''-phosphate-modified cytochromes c by cytochrome c oxidase.

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.     

Interaction of detergents with cytochrome c oxidase.

The binding of ionic and nonionic, nondenaturing detergents to cytochrome c oxidase has been examined. All bind and displace part but not all of the phospholipid that is associated with the enzyme after isolation. From 6 to 10 phospholipid molecules, depending on the detergent used, do not exchange and these are mostly diphosphatidylglycerol molecules as first shown by Awasthi et al. ((1971) Biochim. Biophys. Acta 226, 42). The binding of Triton X-100 and deoxycholate to the cytochrome c oxidase complex has been studied in detail. Both bind to the enzyme above their critical micelle concentrations: Triton X-100 in the amount of 180 +/- 10 molecules per complex and deoxycholate in the amount of 80 +/- 4 molecules per complex. In nonionic detergents, cytochrome c oxidase exists as a dimer (4 heme complex). The enzyme is dissociated into the monomer or heme aa3 complex by delipidation in bile salts. Activity measurements in different detergents suggest that cytochrome c oxidase requires a flexible, hydrophobic environment for maximal activity and that the dimer or 4 heme complex may be the active species.     

Cytochemistry of cytochrome oxidase in the cytoplasmic and intracytoplasmic membranes of Azotobacter vinelandii

Vegetative cells of Azotobacter vinelandii contain a system of intracytoplasmic membranes in the form of numerous internal vesicles. The three-dimensional morphology of these internal vesicles was established by an examination of stereopair electron micrographs of negatively stained cells. The vesicles assumed a variety of forms ranging from nearly spherical units to short, curved tubules. These structures were found at the periphery of the cytoplasm, subjacent to the cytoplasmic membrane. Large flattened cisternae were also present in some cells. The amount of intracytoplasmic membrane varied widely even among individual cells from the same culture. The total surface area of the intracytoplasmic membranes was greater than that of the cytoplasmic membrane in many cells. To assess the possible association of cytochrome oxidase activity with the intracytoplasmic membranes, enzyme localization experiments were conducted with the cytochemical substrate 3,3'-diaminobenzidine. The results showed that a cyanide-sensitive cytochrome oxidase activity is located at the intracytoplasmic membrane. The quantity of cytochrome oxidase activity present in the internal membranes is probably less than that present in the cytoplasmic membrane.     

Modulation of cytochrome oxidase activity by inorganic and organic phosphate.

The activity of cytochrome oxidase reconstituted into phospholipid vesicles has been studied as a function of orthophosphate, ATP and inositol hexakisphosphate concentrations. The respiratory-control ratio was found to be quite sensitive to these compounds and was inversely related to the anion concentration. This effect is related to a phosphate-dependent decrease in the rate constant for ferrocytochrome c oxidation observed in the presence of ionophores. The data cannot be interpreted simply on the basis of ionic strength, which is known to limit cytochrome c binding to cytochrome oxidase, since cytochrome oxidase-containing vesicles responded differently to phosphate depending on the energization state of the phospholipid membrane.     

Purification of a cytochrome aa3 terminal oxidase from protoplast membrane vesicles of Micrococcus luteus

Abstract A cytochrome aa₃ terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 1.9.3.1) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and n-dodecyl-β-d-maltoside and purified by ion-exchange chromatography using poly-L-lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme contains 12.1 nmol of heme A (mg protein)⁻¹ and exhibits absorption maxima at 424 nm and 598 nm in the oxidized state and at 442 nm and 599 nm in the reduced state. The CO-difference spectrum shows peaks at 428 and 590 nm which is indicative of heme a ₃, furthermore oxygen consumption was found to be sensitive to cyanide.     

The cytochrome d complex is a coupling site in the aerobic respiratory chain of Escherichia coli

The cytochrome d complex from Escherichia coli has been reconstituted in proteoliposomes. Previous studies have shown that the enzyme rapidly oxidizes ubiquinol-8 within the bilayer as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane potential across the vesicle bilayer. In this work, the proton pumping activity of the cytochrome in the reconstituted vesicles is examined. Ubiquinol-1 oxidase activity is shown to be accompanied by the net alkalinization of the interior space of the reconstituted vesicles and by the release of protons in the external volume. H+/O ratios varying from 0.6 to 1.2 were measured in different preparations, by the oxygen pulse technique. Antibodies which bind specifically to subunit I (cytochrome b558) of the 2-subunit oxidase were used to estimate the topology of the reconstituted oxidase in the vesicles. It was concluded that 70-85% of the molecules were oriented with subunit I facing the outside and that this population of molecules is responsible for the observed proton release. Correction for the fraction of the oxidase which pumps protons into the vesicle interior yields an estimate of H+/O = 1.7 +/- 0.2. It is proposed that the enzyme does not function as an actual proton pump, but that the enzyme oxidizes ubiquinol and reduces oxygen (to water) on opposite faces of the membrane. Hence, scalar chemistry would yield H+/O = 2 and an electrogenic reaction by virtue of the transmembrane electron transfer between the proposed active sites.     

Lipid and subunit III depleted cytochrome c oxidase purified by horse cytochrome c affinity chromatography in lauryl maltoside

Cytochrome oxidase is purified from rat liver and beef heart by affinity chromatography on a matrix of horse cytochrome c-Sepharose 4B. The success of this procedure, which employs a matrix previously found ineffective with beef or yeast oxidase, is attributed to thorough dispersion of the enzyme with nonionic detergent and a low density of cross-linking between the lysine residues of cytochrome c and the cyanogen bromide activated Sepharose. Beef heart oxidase is purified in one step from mitochondrial membranes solubilized with lauryl maltoside, yielding an enzyme of purity comparable to that obtained on a yeast cytochrome c matrix [Azzi, A., Bill, K., & Broger, C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2447-2450]. Rat liver oxidase is prepared by hydroxyapatite and horse cytochrome c affinity chromatography in lauryl maltoside, yielding enzyme of high purity (12.5-13.5 nmol of heme a/mg of protein), high activity (TN = 270-400 s-1), and very low lipid content (1 mol of DPG and 1 mol of PI per mol of aa3). The activity of the enzyme is characterized by two kinetic phases, and electron transfer can be stimulated to maximal rates as high as 650 s-1 when supplemented with asolectin vesicles. The rat liver oxidase purified by this method does not contain the polypeptide designated as subunit III. Comparisons of the kinetic behavior of the enzyme in intact membranes, solubilized membranes, and the purified delipidated form reveal complex changes in kinetic parameters accompanying the changes in state and assay conditions, but do not support previous suggestions that subunit III is a critical factor in the binding of cytochrome c at the high-affinity site on oxidase or that cardiolipin is essential for the low-affinity interaction of cytochrome c. The purified rat liver oxidase retains the ability to exhibit respiratory control when reconstituted into phospholipid vesicles, providing definitive evidence that subunit III is not solely responsible for the ability of cytochrome oxidase to produce or respond to a membrane potential or proton gradient.     

Variations on the "dilution" method for reconstituting cytochrome c oxidase into membrane vesicles

A method for the rapid incorporation of cytochrome c oxidase into membranes has been developed. This method essentially consists of obtaining a preparation of the enzyme in which it is isolated and then dissolving it in a medium containing 0.5% of the detergent Tween 20, which gives a final concentration of 0.0125% after reconstitution. These studies revealed an optimal ratio of 1 microgram of enzyme to 5 mg of phospholipids. A similar optimal ratio was found when the amount of protein was varied. The optimum temperature was found to be 30 degrees C. Without a peak value being reached, it was found that the best reconstitution was obtained at pH 7.0-8.0. When measurements were performed either with a fluorescent cyanine (DiSC3) or by the uptake of tetraphenylphosphonium, it was found that the enzyme, with cytochrome c added to the outside, was capable of generating a membrane potential that was negative inside. Using the same procedure, the enzyme could also be reconstituted into vesicles of yeast plasma membrane. The procedure, then, seems adequate for incorporating cytochrome c oxidase into different kinds of membrane vesicles.     

Proton translocation by a native and subunit III-depleted cytochrome c oxidase reconstituted into phospholipid vesicles. Use of fluorescein-phosphatidylethanolamine as an intravesicular pH indicator

The existence of a proton pump associated with bovine cytochrome c oxidase (EC 1.9.3.1) has over the last few years been a matter of considerable dispute. In an attempt to resolve some of the problems with the measuring system we have synthesized fluorescein-phosphatidylethanolamine which when reconstituted with cytochrome c oxidase into phospholipid vesicles provided a reliable indicator of the intravesicular pH. It was observed that cytochrome c oxidase catalyzed the abstraction of almost 2 protons from the intravesicular medium/molecule of ferrocytochrome c oxidized. In parallel experiments whereby the extravesicular pH was measured with an electrode it was found that the enzyme appeared to be responsible for the appearance of almost 1.0 proton/molecule of ferrocytochrome c oxidized. Taken together these data unequivocally demonstrate that cytochrome c oxidase behaves as a proton pump. Furthermore, the other proton which was abstracted is believed to be used for the process of the reduction of oxygen. Similar experiments were performed with a cytochrome c oxidase preparation which was devoid of subunit III. Under these circumstances the enzyme appeared to be unable to translocate protons across the vesicular membrane but was competent to abstract protons from the intravesicular medium for the reduction of oxygen.     

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.     

Ca2+-dependent and independent mitochondrial damage in HepG2 cells that overexpress CYP2E1

The effect of detergents on electron and proton transfer in bovine cytochrome c oxidase was studied using steady-state and transient-state methods. Cytochrome c oxidase in lauryl maltoside has high maximal turnover (TN(max)=400 s(-1)), whereas activity is low (TN(max)=10 s(-1)) in Triton X-100. Single turnover studies of intramolecular electron transfer show similar rates in either detergent. Transient proton uptake experiments show the oxidase in lauryl maltoside consumes 1.8+/-0.3 H(+)/aa(3) during either partial reduction of the oxidase or reaction of fully reduced enzyme with O(2). However, the oxidase in Triton X-100 consumes 2.6+/-0.4 H(+)/aa(3) during partial reduction and 1.0+/-0.2 H(+)/aa(3) in the O(2) reaction. Absorption spectra recorded during turnover show that the enzyme undergoes activation in lauryl maltoside, but does not activate in Triton X-100. We propose that cytochrome c oxidase in different detergents allows access to different sites of protonation, which in turn influences steady-state activity.     

Effect of changing the detergent bound to bovine cytochrome c oxidase upon its individual electron-transfer steps

The influence of the detergent environment upon individual electron-transfer rates of cytochrome c oxidase was investigated by stopped-flow spectrophotometry. The effects of three detergents were studied: lauryl maltoside, which supports a high turnover number (TN = 350 s-1), n-dodecyl octaethylene glycol monoether (C12E8), which supports an intermediate TN (150 s-1), and Triton X-100 in which oxidase is nearly inactive (TN = 2-3 s-1). Under limited turnover conditions (cytochrome c:cytochrome c oxidase ratio = 1:1 to 8:1), the rate of oxidation of cytochrome c was measured and compared with the fast reduction of cytochrome a and its relatively slow reoxidation. Two reducing equivalents of cytochrome c were rapidly oxidized in a burst phase; the remaining two to six equivalents were oxidized more slowly, concurrent with the reoxidation of cytochrome a; i.e., the percent reduced cytochrome a reflects the percent reduced cytochrome c. With the resting enzyme, the bimolecular reaction between reduced cytochrome c and cytochrome a was rapid, was insensitive to the detergent environment, and was not the rate-limiting step in the presence of any detergent. The rate of internal electron transfer from cytochrome a to cytochrome a3 in the resting enzyme was slow and only slightly affected by the detergent environment: 1.0-1.1 s-1 in Triton X-100, 5-7 s-1 in C12E8, and 5-12 s-1 in lauryl maltoside. With the pulsed enzyme, the intramolecular electron transfer between cytochrome a and cytochrome a3 increased 4-5-fold in the lauryl maltoside enzyme but did not increase in the Triton X-100 enzyme (intermediate values were obtained with the C12E8 enzyme). We conclude that cytochrome c oxidase acquires the pulsed conformation only in those detergents that support high TN's, e.g., lauryl maltoside and C12E8, but it is locked in the resting conformation in those detergents which result in low TN's, e.g., Triton X-100.     

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.     

Direct observation of release of cytochrome c from lipid-encapsulated protein by peroxide and superoxide: a possible mechanism for drug-induced apoptosis

Release of cytochrome c from inside lipid vesicles and from inside proteoliposomes formed by cytochrome c oxidase has been studied by spectrophotometric methods. The protein encapsulated inside vesicles did not form complex with sodium azide solution added externally. Both hydrogen peroxide and superoxide were found to cause release of cytochrome c from the lipid encapsulated protein, which was detected from the distinct spectral changes due to the formation of the azide complex of cytochrome c in the solution. Cytochrome c encapsulated inside proteoliposomes containing cytochrome c oxidase (CcO) did not release the cytochrome c during enzymatic turnover of CcO. The anticancer drug, doxorubicin, was found to inhibit the biochemical function of cytochrome c oxidase and release of cytochrome c was observed from the proteoliposome encapsulating the protein during the enzymatic turnover in the presence of doxorubicin. The results indicated that the inhibition of enzymatic activity by doxorubicin possibly leads to the formation of reactive oxygen species, which induce the release of cytochrome c from inside to outside of the membrane.     

Kinetic characterization of cytochrome c oxidase from Bacillus subtilis

Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.