F1F0-ATPase from Escherichia coli with mutant F0 subunits. Partial purification and immunoprecipitation of F1F0 complexes

Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed.     

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

The F₁F₀ complex of Paracoccus denitrificans (PdF₁F₀) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF₁F₀-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem. 267, 993–1000) but its effect on the PdF₁F₀-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and ³²Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K _0.5 for Mg²⁺-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC₅₀ of Mg²⁺-ADP for inhibition of the PdF₁F₀-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF₁F₀ complex. These effects are not due to membrane or PdF₁F₀ uncoupling. Kinetic and structural modifications that could account for these results are discussed.     

Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0

Monoclonal and polyclonal antibodies directed against peptides of F₁-ATPase or F₁F₀-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti- monoclonal antibodies has shown asymmetry of in the complex as already shown for . In addition, the involvement of with respect to nucleotide site cooperativity has been detected. Finally, the formation of F₁F₀-antibody complexes of various masses, seems to exclude the functional rotation of F₁ around F₀ during catalysis.Abbreviations IF₁ natural protein inhibitor of the ATPase-ATP synthase - OSCP oligomycin sensitivity-conferring protein - DCCD dicyclohexylcarbodiimide - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoreses - F₁ F₁-ATPase, coupling factor F₁ of ATPase - F₁F₀ F₁F₀-ATP synthase, ATPase-ATP synthase complex     

Torque generation and utilization in motor enzyme F0F1-ATP synthase: half-torque F1 with short-sized pushrod helix and reduced ATP Synthesis by half-torque F0F1

ATP synthase (F(0)F(1)) is made of two motors, a proton-driven motor (F(0)) and an ATP-driven motor (F(1)), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F(1), the central γ subunit rotates inside the α(3)β(3) ring. Here we report structural features of F(1) responsible for torque generation and the catalytic ability of the low-torque F(0)F(1). (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F(1)s, termed F(1)(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid "pushrod" to push the rotor γ subunit, but the short helix in F(1)(1/2)s would fail to accomplish this task. (ii) Three different half-torque F(0)F(1)(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F(0)F(1)(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F(0)F(1)(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants.     

Sequences of alamethicins F30 and F50 reconsidered and reconciled.

From the culture broth of the mould Trichoderma viride, strain NRRL 3199, a microheterogeneous mixture of the membrane active 20-residue peptaibol alamethicin (ALM) could be isolated. ALMs were isolated by XAD-2 column chromatography and separated by silica gel chromatography and trichloromethane/MeOH gradient elution into an acidic and neutral group of peptides, named ALM F30 and ALM F50, respectively, according to their 100 Rf on TLC. Peptides ALM F50 were separated by semi-preparative and analytical HPLC and subjected to ESI-MS. Ten sequences of ALM F30 and their relative quantities could be determined. The major peptides ALM F30/3 (46%) and ALM F30/7 (40%), distinguished by Aib/Ala exchange in position 6, correspond to sequences described as ALM I and II occurring in the original alamethicin from Upjohn Company. Analogously, 13 sequences of the neutral peptide mixture named ALM F50 could be determined. The major peptide ALM F50/5 (75%) and the minor peptide ALM F50/7 (10%) are distinguished from ALM F30/3 and ALM F30/7 by having Gln17 in place of Glu17, the latter occurring in the F30 group. Notably. currently commercially available alamethicins (Fluka, Sigma) represent microheterogeneous mixtures of the neutral ALM F50 peptides with trace amounts of acidic ALM F30 peptides.     

Investigation of DNA complexes with histones F3 and F3+F2a2]

Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA.     

E2F7 and E2F8 keep the E2F family in balance

An article by Li and colleagues (in this issue of Developmental Cell) shows that the atypical E2Fs, E2F7 and E2F8, are critical for mouse development. One of the important functions of these family members stems from a negative feedback loop in which E2F7 and E2F8 limit the expression of E2F1 and prevent E2F1-dependent apoptosis.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.     

Synthesis and biological activities of leukotriene F4 and leukotriene F4 sulfone

Leukotriene F4 (LTF4) and LTF4 sulfone have been synthesized and their biological activities determined in the guinea pig. In vitro LTF4 displayed comparable activity to LTD4 on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF4 induced a bronchoconstriction (ED50 16 micrograms Kg-1) which was blocked by indomethacin and FPL-55712 and was 50-100 X less potent than LTD4 in this assay. LTF4 sulfone was approximately 2-5 times less active than LTF4 in vitro and in vivo. When injected into guinea pig skin with PGE2 (100 ng); LTF4 and LTF4 sulfone (10-1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE4 sulfone = LTD4 = LTD4 sulfone greater than LTE4 greater than LTF4 = LTF4 sulfone.     

Enzymatic deglycosylation of thyroid-stimulating hormone with peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F

We investigated the ability of two enzymes, peptide N-glycosidase F (PNGase F) and endo-beta-N-acetylglucosaminidase F (Endo F), to deglycosylate microgram quantities of bovine TSH and its subunits under nondenaturing conditions. One oligosaccharide chain could be selectively removed from the alpha subunit by PNGase F, and all the oligosaccharide chains from both subunits could be removed by Endo F. These methods of enzymatic deglycosylation should permit study of the functional role of each N-linked carbohydrate chain of various glycoprotein hormones.     

Sialic acid in F. tularensis and F. novicida antigens]

Sialic acid was revealed in the antigens of virulent (503/830 and Schu) and vaccine (Schu-attenuated and 15-reduced) strains of F. tularensis and also in the antigens of F. novicida isolated by the treatment with trichloracetic acid, by the thiobarbiturate method of Warren. Its content depended on the culture virulence. Sialic acid was absent in the antigens of avirulent strains of the causative agent of tularemia (503-attenuated, 15-attenuated and 21/400), but, in difference from the virulent strains, there was revealed 2-desoxyribose on account of the presence of DNA. Serological activity of F. tularensis antigen decreased only 15 times in the antibody neutralization reaction after a complete release of sialic acid as a result of hydrolysis of the preparation in 0.1 N, H2SO4 at 80 degrees C for one hour, i.e. it persisted at a sufficiently high level. On the basis of investigations carried out it can be admitted that there existed a chemical association between the sialic acid and other composites in the polysaccharide of the virulent and the vaccine strains of F. tularensis, but that this acid played no significant role in the manifestation of specific properties by the antigen.     

Origin of the F685 and F695 fluorescence in Photosystem II

The emission spectra of CP47-RC and core complexes of Photosystem II (PS II) were measured at different temperatures and excitation wavelengths in order to establish the origin of the emission and the role of the core antenna in the energy transfer and charge separation processes in PS II. Both types of particles reveal strong dependences of spectral shape and yield on temperature. The results indicate that the well-known F-695 emission at 77 K arises from excitations that are trapped on a red-absorbing CP47 chlorophyll, whereas the F-685 nm emission at 77 K arises from excitations that are transferred slowly from 683 nm states in CP47 and CP43 to the RC, where they are trapped by charge separation. We conclude that F-695 at 77 K originates from the low-energy part of the inhomogeneous distribution of the 690 nm absorbing chlorophyll of CP47, while at 4 K the fluorescence originates from the complete distribution of the 690 nm chlorophyll of CP47 and from the low-energy part of the inhomogeneous distribution of one or more CP43 chlorophylls.     

Inhibition of yeast mitochondrial F1-ATPase, F0F1-ATPase and submitochondrial particles by rhodamines and ethidium bromide

ATP hydrolysis by F1-ATPase is strongly inhibited by cationic rhodamines; neutral rhodamines are very poor inhibitors. Rhodamine 6G is a noncompetitive inhibitor of purified F0F1-ATPase and submitochondrial particles, however, an uncompetitive inhibitor of F1-ATPase (KI approximately equal to 2.4 microM for all three enzyme forms). Ethidium bromide is a noncompetitive inhibitor of F0F1-ATPase, submitochondrial particles and also F1-ATPase (KI approximately equal to 270 microM). Neither of the inhibitors affects the negative cooperativity (nH approximately equal to 0.7). The non-identical binding sites for rhodamine 6G and ethidium bromide are located on the F1-moiety and are topologically distinct from the catalytic site. Binding of the inhibitors prevents the conformational changes essential for energy transduction. It is concluded that the inhibitor binding sites are involved in proton translocation. In F1-ATPase, binding of MgATP at a catalytic site causes conformational changes, which allosterically induce the correct structure of the rhodamine 6G binding site. In F0F1-ATPase, this conformation of the F1-moiety exists a priori, due to allosteric interactions with F0-subunits. The binding site for ethidium bromide on F1-ATPase does not require substrate binding at the catalytic site and is not affected by F0F1-subunit interactions.     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

Thermal inactivation of electron-transport functions and F0F1-ATPase activities

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)