Molecular dynamics simulations of the rotary motor F(0) under external electric fields across the membrane.

The membrane-bound component F(0), which is a major component of the F(0)F(1)-ATP synthase, works as a rotary motor and plays a central role in driving the F(1) component to transform chemiosmotic energy into ATP synthesis. We conducted molecular dynamics simulations of b(2)-free F(0) in a 1-palmitoyl-2-oleoyl-phosphatidylcholine lipid bilayer for tens of nanoseconds with two different protonation states of the cAsp-61 residue at the interface of the a-c complex in the absence of electric fields and under electric fields of +/-0.03 V/nm across the membrane. To our surprise, we observed that the upper half of the N-terminal helix of the c(1) subunit rotated about its axis clockwise by 30 degrees . An energetic analysis revealed that the electrostatic repulsion between this N-terminal helix and subunit c(12) was a major contributor to the observed rotation. A correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. The deuterium order parameter (S(CD)) profile calculated by averaging all the lipids in the F(0)-bound bilayer was not very different from that of the pure bilayer system, in agreement with recent (2)H solid-state NMR experiments. However, by delineating the lipid properties according to their vicinity to F(0), we found that the S(CD) profiles of different lipid shells were prominently different. Lipids close to F(0) formed a more ordered structure. Similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. The lipids in the proximity of F(0) exhibited very significantly reduced diffusional motion. The numerical value of S(CD) was anticorrelated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. Our findings will help elucidate the dynamics of F(0) depending on the protonation state and electric field, and may also shed some light on the interactions between the motor F(0) and its surrounding lipids under physiological conditions, which could help to rationalize its extraordinary energy conversion efficiency.

Using F0F1-ATPase motors as micro-mixers accelerates thrombolysis.

We have developed a novel micro-mixer using a biological molecular ATP motor. The micro-mixer was constructed from arrays of chromatophore-embedded delta-free F(0)F(1)-ATPases, where the delta-free F(1) part acted as a rotator to mix solutions, and the F(0) part was driven by light. Confocal microscope studies indicated that the micro-mixer did not touch directly on the fibrin labeled with FITC. The nanomechanical force generated by the motor induced drug movement in the solution and accelerated the fibrinolysis process. All results strongly suggest that the micro-mixers generated a nanomechanical force which accelerated the fibrinolysis process in the presence of lower concentrations of lumbrokinase.

Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF1): the effect on the CF1-ATPase activity.

Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19-24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[beta-32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the beta subunit of the CF1 and since there are probably three beta subunits per CF1, these results indicate strong cooperativity among beta subunits and between the site of tightly bound nucleotides and the catalytic sites.

Functional molecular masses of vacuolar membrane H+-ATPase from Saccharomyces cerevisiae as studied by radiation inactivation analysis.

The functional molecular masses of the vacuolar membrane H+-ATPase in Saccharomyces cerevisiae under two kinetic conditions for ATP hydrolysis were measured by radiation inactivation. When vacuolar membrane vesicles were exposed to gamma-rays from 60Co, the activities catalyzing a single-cycle and multi-cycles of ATP hydrolysis both decreased as single-exponential functions of the radiation dosage. By applying the target theory, the functional molecular masses for single- and multi-cycle hydrolyses of ATP were determined to be approx. 0.9-1.1 X 10(5) and 4.1-5.3 X 10(5) Da, respectively. N,N'-Dicyclohexylcarbodiimide (DCCD) did not inhibit the former reaction but strongly inhibited the latter. It is suggested that the ATPase with a minimal composite of subunits a and b, in which subunit c is not necessarily involved operationally, can catalyze single-cycle hydrolysis of ATP, whereas for multi-cycle hydrolysis of ATP, the ATPase requires a properly organized oligomeric structure with subunits a-c, which may direct a positive cooperative mechanism of ATP hydrolysis and coupled H+ translocation in a DCCD-sensitive manner.

2,8-Diazido-ATP--a short-length bifunctional photoaffinity label for photoaffinity cross-linking of a stable F1 in ATP synthase (from thermophilic bacteria PS3).

To demonstrate the direct interfacial position of nucleotide binding sites between subunits of proteins we have synthesized the bifunctional photoaffinity label 2,8-diazidoadenosine 5'-triphosphate (2,8-DiN3ATP). UV irradiation of the F1-ATPase (TF1) from the thermophilic bacterium PS3 in the presence of 2,8-DiN3ATP results in a nucleotide-dependent inactivation of the enzyme and in a nucleotide-dependent formation of alpha-beta crosslinks. The results confirm an interfacial localization of all the nucleotide binding sites on TF1.

ADP tethered to tyrosine-beta 345 at the catalytic site of the bovine heart F1-ATPase is converted to tethered AMP by Mg(2+)-dependent hydrolysis when the enzyme is photoinactivated with 2-N3-ADP.

Comparison of profiles of radioactive peptides resolved by HPLC from tryptic digests of the bovine heart F1-ATPase depleted of nucleotides (nd-MF1) which had been photoinactivated with 2-N3-[beta-32P]ADP, on the one hand, and 2-[8-3H]ADP, on the other, shows that the beta phosphate of ADP tethered to tyrosine-beta 345 is slowly hydrolyzed in the presence of Mg2+. When nd-MF1 was photoinactivated with 2-N3-[8-3H]ADP in the absence of Mg2+, hydrolysis of the beta phosphate from ADP tethered to tyrosine-beta 345 was not observed. Subsequent addition of Mg2+ initiated conversion of ADP tethered to tyrosine-beta 345 to tethered AMP suggesting that functional groups at the catalytic site participate in the hydrolytic reaction.

3'-Arylazido-beta-alanyl-2-azido ATP, a cross-linking photoaffinity label for F1ATPases.

The synthesis of the 3'-arylazido-2-azido ATP derivative 3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino]propionyl)2-azido-adenosine 5'-triphosphate (2,3'-DiN3ATP) is described. The bifunctional photoreactive ATP analog is characterized spectroscopically. Photoaffinity labeling of F1ATPase from Micrococcus luteus by this analog results in the inactivation of the enzyme and in the formation of higher molecular weight cross-links, composed of alpha- and beta-subunits.

[Electron paramagnetic resonance of the F1-H+-ATPase complex and adenosine triphosphate synthesis after its irradiation with visible light]. / Elektronnyi paramagnitnyi rezonans kompleksa F1-H+-ATPazy i sintez adenozintrifosfata pri ego osveshchenii vidimym svetom.

Under F1-ATPase irradiation by visible light two characteristic features of flavin main activities were detected: I/ESR signal g = 2.00 appearance which was in favor of flavin photochemical electron reduction and 2/photostimulated O2 consumption by F1-ATPase. The dependence of ESR signal g = 2.00 intensity on visible light wavelength completely coincided with the same dependence obtained for proteinless model riboflavin + ADP. Flavin localization in proximity of ADP bound in the enzyme active site was suggested. Under F1-ATPase irradiation by light lambda greater than 350 nm ATP synthesis was obtained similar to the proteinless model riboflavin + ADP + Pinorg. In this model endogenous flavin was suggested to serve as a photosensitizer, its photoexcitement being a model of dark energization of F1-ATPase in oxidative phosphorylation.

[Energizing of F1-ATPase after irradiation with visible light]. / Energizatsiia F1-ATFazy pri obluchenii vidimym svetom.

It was shown that ATP synthesis by F1-ATPase sensitized by visible light was combined with oxidation of SH groups and decrease of initial flavin fluorescence in the F1-ATPase preparation. It was suggested that it was an endogenous flavin group that regulated redox transitions between SH-S-S groups which was essential for the catalysis in vivo.

Harmonic response of cellular membrane pumps to low frequency electric fields.

We report on harmonic generation by budding yeast cells in response to a sinusoidal electric field, which is seen to be minimal when the field amplitude is less than a threshold value. Surprisingly, sodium metavanadate, an inhibitor of P-type ATPases reportedly responsible for nonlinear response in yeast, reduces the threshold field amplitude, increasing harmonic generation at low amplitudes while reducing it at large amplitudes, whereas the addition of glucose dramatically increases the production of even harmonics. Finally, a simple model is proposed to interpret the observed behavior.

Radiation-inactivation analysis of vacuolar H(+)-ATPase and H(+)-pyrophosphatase from Beta vulgaris L. Functional sizes for substrate hydrolysis and for H+ transport.

The functional sizes of the vacuolar H(+)-ATPase (V-ATPase; EC 3.6.1.34) and H(+)-pyrophosphatase (PPase; EC 3.6.1.1) from vacuolar membranes of red beet (Beta vulgaris L.) were estimated by radiation inactivation, both for substrate hydrolysis and for H+ transport. For the V-ATPase, the radiation-inactivation size for H+ transport was 446 (403-497) kDa and that for ATP hydrolysis was 394 (359-435) kDa. The low values of both of these estimates suggest that not all subunits which may co-purify with V-ATPases are required for either hydrolysis or transport. For the PPase, the radiation-inactivation size for hydrolysis was 91 (82-103) kDa, suggesting that the minimum functional unit for hydrolysis is the 81 kDa monomer. In contrast to the V-ATPase, the PPase gave a radiation-inactivation size for transport which was 3-4-fold larger than that for hydrolysis (two estimates for transport gave 307 and 350 kDa), indicating that a single catalytic subunit is insufficient for transport activity.

Catalytic and regulatory effects of light intensity on chloroplast ATP synthase.

The incorporation of water oxygens into ATP made by photophosphorylation is known to be increased markedly when either Pi or ADP concentration is lowered. The present studies show a similar increase in oxygen exchange when light intensity is lowered even with ample ADP and Pi present. The number of reversals of bound ATP formation prior to release increases about 1 to about 27 in the presence of dithiothreitol and to 5 in its absence. The equilibrium of the bound reactants still favors ATP at low light intensity, as shown by measurement of the amount of bound ATP rapidly labeled from [32P]Pi during steady-state photophosphorylation. Changes observed in the interconversion rate in the absence of added thiol are likely involved in the regulation of the dark ATPase activity in the chloroplast. The interconversion rate of bound ATP to bound ADP and Pi in the presence of thiol is about the same at low and high light intensities. This rate of bound ATP formation is not sufficient, however, to account for the maximum rate of photophosphorylation. Thus, when adequate protonmotive force is present, the rate of conversion of bound ADP and Pi to bound ATP, and possibly that of bound ATP to bound ADP and Pi, must be increased, with proton translocation being completed only when bound ATP is present to be released. These observations are consistent with the predictions of the binding change mechanism with sequential participation of catalytic sites and are accommodated by a simplified general scheme for the binding change mechanism that is presented here.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity cross-linking of the coupling factor 1 from Micrococcus luteus by 3'-arylazido-8-azido-ATP.

The vicinity of nucleotide binding sites and the mechanism of ATP synthesis/hydrolysis have been studied with the bifunctional photosensitive ATP analog 3'-arylazido-8-azido-ATP. 3'-Arylazido-8-azido-ATP is hydrolyzed by the F1-ATPase from Micrococcus luteus in the absence of ultraviolet light. Irradiation, by ultraviolet light, of F1-ATPase in the presence of 3'-arylazido-8-azido-ATP results in the specific formation of cross-links between alpha and beta subunits. The results suggest that a hydrolytic nucleotide binding site is located on a beta subunit at or near an alpha subunit, probably at the interface between these subunits. Such a constellation would permit direct subunit-subunit interactions during ATP synthesis/hydrolysis.

The effect of low-intensity laser irradiation on the hydrolytic activity of vacuolar membrane proton pumps.

The effect of low-intensity irradiation by a helium-neon laser on the hydrolytic activity of the vacuolar membrane proton pumps has been studied. The maximum effect was found for the 3 min irradiation from a close distance (0.3 m); moreover, the PPase activity increased by 33%, whereas the ATPase activity was inhibited by 44%. The effect described is suggested to be due to different conformations of the enzymes in the membrane and their different physiological roles.

Differential effect of thiol oxidants on the chloroplast H+-ATPase in the light and in the dark.

The effect of thiol oxidants on the light-activated H+-ATPase has been studied in freshly broken and intact chloroplasts. The following observations were made: (i) in chloroplasts which are osmotically shocked after light activation, ferricyanide stimulates the deactivation of the enzyme in the dark, but has little effect in the light; (ii) similarly, o-iodosobenzoate is a most efficient deactivator of the ATPase in intact chloroplasts in the dark but not in the light; (iii) the activated ATPase becomes sensitive to oxidants in the light upon the addition of an uncoupler; (iv) the oxidant-induced deactivation in the dark dominates the stabilizing effect of pyrophosphate or ADP plus Mg2+; (v) full deactivation of the ATPase by dark adaptation or by oxidants does not affect the rate of photophosphorylation under saturating conditions. A model is suggested in which two kinds of conformational changes are involved in the regulation of the ATPase: those induced by the trans-membrane-proton gradient and those by oxidation-reduction of the enzyme. These changes result in the preferential interaction with thiol reductants in the light but with thiol oxidants in the dark.

Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle.

Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.

Inactivation of mitochondrial ATPase by ultraviolet light.

The present work describes experiments that show that far-ultraviolet irradiation induce the inhibition of ATPase activity in both membrane-bound and soluble F1. It was also found that ultraviolet light promotes the release of tightly bound adenine nucleotides from F1-ATPase. Experiments carried out with submitochondrial particles indicate that succinate partially protects against these effects of ultraviolet light. Titration of sulfhydryl groups in both irradiated submitochondrial particles and soluble F1-ATPase indicates that a conformational change induced by photochemical modifications of amino acid residues appears involved in the inactivation of the enzyme. Finally, experiments are described which show that the tyrosine residue located in the active site of F1-ATPase is modified by ultraviolet irradiation.

Regulation of the chloroplast H+-ATPase by light. The involvement of Mg2+ ions.

The involvement of Mg2+ ions in the light-dependent regulation of the chloroplast H+-ATPase was studied in both type C and osmotically shocked type A chloroplasts. The following results were obtained. ATPase activity measured under dark, partially uncoupling conditions, following light activation with dithiothreitol and pyocyanine, was markedly enhanced by the presence of Mg2+ in the activation stage. This Mg2+ effect required concentrations in the millimolar range, was rather slow (time range of minutes), reversible, rather unspecific and did not involve changes in the affinity to dithiothreitol. Dark deactivation of the ATPase in the absence of substrate was accelerated by Mg2+. The dark effect of Mg2+ also required millimolar concentrations, but was fast (time range of seconds), highly specific for Mg2+, and did not involve thiol oxidation. The major effect of the absence of Mg2+ from the light-activation stage or of its presence in the dark interval between activation and assay was the induction of an 'abnormal' sensitivity to uncouplers: after these treatments ATP hydrolysis was not stimulated but rather inhibited by NH4Cl or other uncouplers. The pretreatments in the light without Mg2+ or dark with Mg2+ did not affect the membrane proton permeability, nor the proton pumping coupled to ATPase activity. The results are discussed in terms of Mg2+-dependent regulation of the enzyme complex at the level of subunit interaction and its effect on the affinity to protons.

Functional size analysis of F-ATPase from Escherichia coli by radiation inactivation.

A radiation inactivation technique was employed to determine the functional size of adenosine triphosphatase from Escherichia coli (EF0EF1-ATPase). Functional units of the membrane-bound and the soluble ATPases were estimated to be 300 +/- 39 and 295 +/- 32 kDa, respectively. The presence of the free radical scavenger dithiothreitol was crucial in measuring the radiation inactivation size of ATPase. When gramicidin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were added, an increase in the functional mass of membrane-bound ATPase was observed. In contrast, valinomycin and KCl had hardly any effect on the functional size of ATPase. We also determined a functional unit of 355 +/- 33 kDa for proton translocation by a fluorescence quenching technique. A reconstitution study using irradiated coupling factor 1 (EF1)-depleted membrane revealed that the functional mass of the proton channel was 96 +/- 11 kDa. A similar functional size for ATP-Pi exchange and ATP hydrolysis implies that both reactions might utilize identical machinery. Furthermore, functional units of soluble EF1 for unisite (nonsteady state) and multisite (steady state) ATP hydrolysis were calculated as 200 +/- 32 and 298 +/- 32 kDa, respectively. A working hypothesis was proposed from radiation inactivation analysis to elucidate the structure and mechanism of F1-ATPase.

Photoinactivation of the F1-ATPase from spinach chloroplasts by dequalinium is accompanied by derivatization of methionine beta183.

In contrast to the F1-ATPases from bovine mitochondria and the thermophilic Bacillus PS3, which are reversibly inhibited by dequalinium in the absence of irradiation, the Mg2+-ATPase activity of heat- or dithiothreitol-activated chloroplast F1 (CF1) from spinach chloroplasts is slightly stimulated by dequalinium. Conversely, dequalinium is a partial inhibitor (maximal inhibition is 85-90%) of the Ca2+-ATPase of CF1 activated by heat, dithiothreitol, or octylglucoside. The Mg2+- and Ca2+-ATPase activities of CF1 respond differently in the presence of lauryl dimethylamine oxide (LDAO) in the assay medium. Whereas the Mg2+-ATPase activity of heat- or dithiothreitol-activated CF1 is stimulated up to 14-fold by increasing concentrations of LDAO, the Ca2+-ATPase is inhibited in a biphasic manner by increasing concentrations of LDAO. In the presence of LDAO, dequalinium does not stimulate the heat-activated Mg2+-ATPase over that promoted by LDAO alone. That dequalinium slightly stimulates Mg2+-ATPase activity although it inhibits Ca2+-ATPase activity can be reconciled by assuming that dequalinium binds to two sites in CF1, a stimulatory site that also binds LDAO and an inhibitory site. By acting as a partial inhibitor of the Mg2+-ATPase activity that it activates, the combined effect of dequalinium is modest stimulation. Irradiation of heat- or dithiothreitol-activated CF1 or the alpha3beta3gamma subcomplex of CF1 in the presence of 12 microM dequalinium led to rapid photoinactivation. ATP and ADP, separately or in combination with Mg2+, protect against photoinactivation. After photoinactivating the alpha3beta3gamma subcomplex of CF1 with [14C]dequalinium, tryptic and peptic digests of the isolated, derivatized beta subunit were fractionated by high performance liquid chromatography. Sequencing of the isolated, radioactive tryptic and peptic peptides revealed that Metbeta183, which is at or near the catalytic site, is derivatized in a single beta subunit when CF1 is photoinactivated with [14C]dequalinium.