Phosphorylation and phosphate-ATP exchange catalyzed by the ATP synthase isolated from Wolinella succinogenes

The ATP synthase, isolated from Wolinella (formerly Vibrio) succinogenes could be fully incorporated into liposomes without significant cleavage of the enzyme or loss of activity. These proteoliposomes, but not the isolated enzyme, catalyzed phosphate-ATP exchange and the phosphorylation of ADP which was driven by an artificially imposed delta mu H across the liposomal membrane. Phosphorylation driven by light was catalyzed by proteoliposomes containing also bacteriorhodopsin. The three activities were similarly sensitive to protonophores or dicyclohexylcarbodiimide. This sensitivity was similar to that of the electron-transport-driven phosphorylation catalyzed by bacterial membrane vesicles. With a delta mu H value 280 mV to drive phosphorylation the turnover number of the enzyme was in the same order of magnitude as that measured in the electron-transport-driven phosphorylation catalyzed by the bacterial membrane. When the delta mu H was below 150 mV, the phosphorylation activity of the incorporated enzyme was two orders of magnitude slower, and was about as fast as light-driven phosphorylation or as the exchange reaction.     

Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.     

Reconstitution in liposomes of the electron-transport chain catalyzing fumarate reduction by formate

Fumarate reduction by formate in Vibrio succinogenes is catalyzed by a membrane-bound electron-transport chain, and is coupled with the phosphorylation of ADP. The electron-transport chain was reconstituted in liposomes from the isolated components. The formate dehydrogenase complex (three different peptides), the fumarate reductase complex (three different peptides) and vitamin K-1 were required for the electron transport. The pathway of the electrons from formate to fumarate in the reconstituted chain was identical with that in the bacterial membrane. Each of the active enzyme complexes in the liposomes participated in the electron transport. This was valid for proteoliposomes with ratios of the contents of the two enzyme complexes ranging between 0.1 and 10. This indicates that vitamin K-1 forms a diffusible pool within the liposomal membrane that allows every quinone molecule to react with each molecule of the two enzyme complexes.     

Voltage dependence of liver gap-junction channels reconstituted into liposomes and incorporated into planar bilayers.

The voltage dependence of rat liver gap junctions was investigated using non-denaturing solubilization and reconstitution of gap-junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single-channel conductance was voltage independent, and its magnitude was 700-1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed-state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage-sensitive conformational changes.     

Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles

Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system.     

Evidence for the Functional Association of Enzyme I and HPr of the Phosphoenolpyruvate-Sugar Phosphotransferase System With the Membrane in Sealed Vesicles of Escherichia coli

Several independent assay procedures were used to estimate the activities of the enzyme constituents of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) in osmotically shocked bacterial membrane vesicles. The soluble enzymes of the system were found to be in association with the membrane by several criteria. Phosphoenolpyruvate-dependent sugar phosphorylation was catalyzed by this membrane-bound enzyme system far more efficiently than by a mixture of the individual enzymes at corresponding concentrations. By contrast, the rates of the phosphoryl exchange reactions catalyzed by enzyme I and the enzyme II complexes were essentially the same for the associated and dissociated forms of the system. Functional association of the PTS-enzyme complex was stabilized by Mg⁺⁺ and phosphoenolpyruvate and could be destroyed by detergent treatment, sonication, or by passage of the vesicle preparation through a French pressure cell. These results lead to the possibility that in the intact bacterial cell the soluble enzymes of the phosphotransferase system exist, in part, as peripheral membrane constituents associated with the integral membrane enzyme II complexes.     

The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase.

Horse kidney brush border membrane proteins were incorporated into phosphatidylcholine vesicles. Structural analysis of proteoliposomes prepared with various lipid:protein ratios showed that: (a) only a few of the proteins present in the crude brush border extract are integrated, (b) all known membrane hydrolases are integrated, and (c) these proteoliposomes are homogeneous vesicles. Papain solubilization of brush border membrane hydrolases, i.e. aminopeptidase M, neutral alpha-glucosidase, gamma-glutamyltransferase and alkaline phosphatase, performed in parallel on native membrane vesicles and proteoliposomes, revealed similar kinetics. Analysis of membrane vesicles and proteoliposomes on sucrose density gradients either without any treatment, or after papain treatment showed that: (a) in proteoliposomes, neutral alpha-glucosidase is associated with radiolabelled phosphatidylcholine, and (b) papain-treated vesicles and proteoliposomes released enzyme activity in the same way. These results suggest that the integration mechanism of brush border membrane proteins may be similar in proteoliposomes and native membrane vesicles. Transport experiments under equilibrium exchange conditions showed that the uptake properties of proteoliposomes are similar to those of brush border membrane vesicles.     

ATP-synthesis by proteoliposomes incorporating Rhodospirillum rubrum F0F1 as measured with firefly luciferase: dependence on delta psi and delta pH

ATP-synthesis catalyzed by proteoliposomes incorporating Rhodospirillum rubrum F0F1 was driven by artificially applied electrochemical proton gradients. The time-course of ATP-synthesis was followed continuously by means of firefly luciferase. Correction methods were developed which allow one to calculate the initial rate of ATP-synthesis from the observed luminescence kinetics. The following results were obtained: (1) ATP-synthesis occurred above a threshold delta mu H+ of 90 mV; this threshold is not imposed by the activation requirement of the enzyme; (2) delta psi and delta pH appear to be equivalent as driving forces for ATP-synthesis if allowance is made for the effect of the electrical capacitance of the liposome membrane on the distribution of K+ at equilibrium; and (3) the highest rate observed so far is 200 mol ATP per mol F0F1 per s.     

Cyclic GMP regulation of the plasma membrane (Ca2+-Mg2+)ATPase in vascular smooth muscle

Plasma membrane (Ca2+-Mg2+)ATPase purified from bovine aortic microsomes by calmodulin affinity chromatography was incorporated into soybean phospholipid liposomes. In the reconstituted proteoliposomes, a protein corresponding to the ATPase was phosphorylated by [gamma-32P]ATP in the presence of cGMP and cGMP-dependent protein kinase. Both the affinity for Ca2+ and the maximum Ca2+ uptake activity by the proteoliposomes were increased by the cGMP-dependent phosphorylation, and there was good parallelism between the Ca2+-uptake rate and the extent of phosphorylation. These results strongly suggest that the Ca2+-transport ATPase of the vascular smooth muscle plasma membrane is regulated through its cGMP-dependent phosphorylation.     

Kinetic study of a phosphoryl exchange reaction between fructose and fructose 1-phosphate catalyzed by the membrane-bound enzyme II of the phosphoenolpyruvate-fructose 1-phosphotransferase system of Bacillus subtilis.

A phosphoryl exchange reaction between fructose 1-phosphate and fructose was found to be catalyzed by a membrane preparation isolated from Bacillus subtilis. The regulation of the biosynthesis of the activity in the wild type as well as in the regulation mutants fruB closely correlates with that of the membrane-bound enzyme II of the phosphoenolpyruvate fructose 1-phosphotransferase system which is known to mediate the transmembrane vectorial phosphorylation of fructose. The computed analysis of the kinetic data shows that the mechanism of the enzyme II is ping-pong, i.e. that a phosphoryl-enzyme intermediate occurs in the reaction. The apparent dissociation constants of the enzyme II/fructose 1-phosphate complex and of the phosphoryl enzyme II/fructose complex are estimated. The value of the standard free energy of the hydrolysis of the bond between the phosphoryl moiety and the enzyme suggests a covalent bonding. This intermediate is assumed to occur in the physiological functioning of the enzyme which utilizes the phosphocarrier protein HPr as phosphoryl donor. The exchange reaction is competitively inhibited by high fructose concentrations: this indicates that the same site of the enzyme binds fructose and fructose 1-phosphate, this site being accessible to fructose on the external side of the membrane when the enzyme is phosphorylated.     

Incorporation of Na,K-ATPase into human erythrocyte membranes using liposomes

A procedure for incorporation of isolated cattle brain Na,K-ATPase into erythrocyte membranes by proteoliposomes has been elaborated. The Na,K-ATPase activity of proteoliposome-treated human erythrocytes containing incorporated Na,K-ATPase does not exceed that of control erythrocytes. In the erythrocyte membrane the incorporated enzyme exists in a functionally active state and retains the vector properties of the Na+-pump. Exogenous ATP stimulates 22Na influx and 86Rb efflux in and from the erythrocytes.     

Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria

The tricarboxylate carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatography on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-saturation constant was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the maximum exchange rate at 25 degrees C reached 2000 mumols/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).     

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.     

Oxidative phosphorylation in Micrococcus denitrificans. I. Preparation and properties of phosphorylating membrane fragments

A procedure was described to prepare stable membrane fragments from aerobically grown cells of Micrococcus denitrificans. This preparation contained flavins, cytochromes b, c, a and o, and catalyzed the synthesis of ATP coupled to the oxidation of NADH and succinate. The P:O ratios were about 1.0 for NADH and 0.4 for succinate oxidation. The electron-transfer pathways responsible for these oxidations were similar to, though not identical with, those of mammalian mitochondria in their construction and sensitivity to inhibitors. Oxidative phosphorylation by the membrane fragments was uncoupled by the usual uncouplers and energy-transfer inhibitors, though 2,4-dinitrophenol was much less effective and higher concentrations of oligomycin and tributyltin chloride were required for complete inhibition as compared with the mitochondrial system. Oleate also caused uncoupling, which was relieved by serum albumin. Treatment with high concentrations of LiCl yielded an essentially uncoupled preparation, but this treatment as well as many other procedures failed to yield soluble coupling factors. Unlike the mitochondrial ATPase activity, ATP hydrolysis by the membrane fragments was inhibited to about 50% by uncouplers and energy-transfer inhibitors. It seems that the bacterial preparation possessed two types of ATPase, one of which was sensitive to these reagents as well as to LiCl treatment and probably to high concentrations of ADP. The advantage of this preparation for the study of the mechanism of oxidative phosphorylation is discussed.     

Dissociation of the sodium-ion-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and reconstitution of the active complex from the isolated subunits

Oxaloacetate decarboxylase was reconstituted from the purified alpha subunit and a Triton X-100 extract of bacterial membranes devoid of this protein. Upon freezing of oxaloacetate decarboxylase in salt solutions, the enzyme was split into subunits and the catalytic activity was abolished. The catalytically active decarboxylase complex was reconstituted by decreasing the salt concentration of the dissociated sample. The conditions for the inactivation were critical for an optimum recovery of catalytically active enzyme during reconstitution, and modest dissociating conditions generally improved the yield of the reconstitutively active decarboxylase. The dissociated enzyme has been separated by chromatography on avidin-Sepharose into two fractions: fraction I, that was not retained on the column, consisted of the beta + gamma subunits, and fraction II consisted of the biotin-containing alpha subunit. Oxaloacetate decarboxylase was reconstituted from a mixture of the isolated alpha and beta + gamma subunits. The Na+ transport activity was recovered, if a mixture of subunits alpha and beta + gamma was incorporated into liposomes, or by a sequential reconstitution, starting with the formation of proteoliposomes with the integral membrane proteins beta + gamma and completed by an attachment of the peripheral subunit alpha.     

A micro-batchwise technique method for rapid reconstitution of functionally active mitochondrial ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers

A method for rapid reconstitution of ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers mitochondria in proteoliposomes is described. The method is based on the well known property of the Amberlite resin to absorb the detergent allowing proteoliposome formation. This has been achieved by a micro-batchwise technique, using a rotating plate stirrer. An evaluation of the optimal conditions, in comparison with the more usual column method is presented. The purified ADP/ATP carrier, incorporated in proteoliposomes by this method, shows a high transport activity and a higher specific activity with respect to proteoliposomes obtained by the column procedure. Furthermore the proteoliposomal preparations are more homogeneous in size, with a diameter ranging from 300 to 350 nm. The method is suitable for the reconstitution of other membrane transport proteins.     

Incorporation of Na+ - Ca2+ antiporter and of (Na+ + K+)-ATPase into liposomes and demonstration of their non-identity

(Na+ + K+)-ATPase was isolated from the grey matter of brain and incorporated into liposomes. Most of the reconstituted enzyme was oriented 'inside-out' with respect to its in vivo orientation and externally added ATP promoted Na+ uptake that was inhibitable by internally trapped ouabain. Using the same proteoliposomes, an Na+ - Ca2+ exchange system was observed as indicated by the following pieces of evidence. (1) The Na+ gradient provided the only readily apparent driving force for acceleration of Ca2+ accumulation into proteoliposomes. (2) The antiporter was specific for Ca2+, high Mg2+ excess did not inhibit Ca2+ antiport. (3) The Na+ efflux was dependent on the extravesicular Ca2+ concentration. (4) The Na+ efflux was not inhibited by tetrodotoxin. The demonstrated Na+ - Ca2+ exchange could not be related to (Na+ + K+)-ATPase protein, since it was not purified with (Na+ + K+)-ATPase, as followed from transport studies with liposomes containing (Na+ + K+)-ATPase of different specific activity. The results strongly indicate that plasma membranes isolated from the grey matter of brain contain an Na+ - Ca2+ exchange system and that the proteoliposomes are suitable for further purification of the carrier molecule.     

Uncoupling Effect of Fatty Acids in Halo- and Alkalotolerant Bacterium Bacillus pseudofirmus FTU

Natural uncouplers of oxidative phosphorylation, long-chain non-esterified fatty acids, cause uncoupling in the alkalo- and halotolerant bacterium Bacillus pseudofirmus FTU. The uncoupling effect in the bacterial cells was manifested as decrease of membrane potential and increase of respiratory activity. The membrane potential decrease was detected only in bacterial cells exhausted by their endogenous substrates. In proteoliposomes containing reconstituted bacterial cytochrome c oxidase, fatty acids caused a "mild" uncoupling effect by reducing membrane potential only at low rate of membrane potential generation. "Free respiration" induced by the "mild" uncouplers, the fatty acids, can be considered as possible mechanism responsible for adaptation of the bacteria to a constantly changed environment.     

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+.     

Incorporation of a bacterial membrane-bound hydrogenase into proteoliposomes.

Membrane-bound nickel-iron hydrogenases from diverse genera of bacteria have been previously characterized and they are closely related. We report the reconstitution of purified Bradyrhizobium japonicum hydrogenase into proteoliposomes by a detergent dialysis method followed by two or three cycles of freeze-thaw. Sedimentation experiments revealed that more than 60% of the H2-uptake activity was particulate when reconstituted into Escherichia coli phospholipids. Sucrose-gradient centrifugation separated hydrogenase activity into two peaks, the less dense of which was phospholipid-associated and turbid, thereby showing successful incorporation. Purified enzyme did not bind to performed phospholipid vesicles, and 1.0 M NaCl failed to remove incorporated hydrogenase. The optimal micellar detergent:phospholipid ratio (rho) value for hydrogenase incorporation was 2.0. Proteoliposomes containing acidic phospholipids were the most effective for incorporation as well as for activity. The artificial electron acceptor specificity was similar for proteoliposomes and for H2-oxidizing membranes from B. japonicum. Proteoliposomes formed under optimal conditions had a broad size distribution centered around 400 nm diameter. Hydrogenase activity in proteoliposomes was partially protected from inactivation by the protein modification reagent diazobenzene sulfonate (DABS) (inactivation t1/2 = 30 min), whereas DABS rapidly inactivated the purified enzyme (t1/2 = 4 min). The latter result indicates protection of a catalytically important site by the phospholipid bilayer. This experimental system should be useful in addressing questions regarding the in vivo situation of bacterial membrane-bound hydrogenases.