The DCCD-binding polypeptide alone is insufficient for proton translocation through F0 in membranes of Escherichia, coli

In contrast to membrane vesicles of wild-type strains which become leaky to protons on removal of the F₁ ATPase, those of the mutant $\text{Escherichia}\text{,}\text{coli}\text{,}\text{N}{}_{\mathrm{I44}}$, which lacks the F₁ ATPase, can maintain a proton gradient. A normal N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide is present in the F₀ portion of the ATPase complex of the mutant. However, the 19000 molecular weight component of F₀ is absent. We conclude that the latter polypeptide, in addition to the DCCD-binding polypeptide, is required for a functional proton channel in F₀.     

Subunit specific antisera to the Escherichia coli ATP synthase: effects on ATPase activity, energy transduction, and enzyme assembly

We obtained antisera to each of the five subunits (α, β, γ, δ, and ?) of the F₁ portion of the proton-translocating ATPase from Escherichia coli (ECF₁). No cross-reaction between the antiserum to a given subunit and any of the other four subunits was observed by Ouchterlony immunodiffusion. The α antiserum reacted only with the denatured α chain. Antibodies to either subunit β or subunit γ inhibited the ATPase activity of the enzyme. The ATPase activity of the holoenzyme in the everted membrane vesicles was just as sensitive as purified ECF₁ to inhibition by the anti-β or anti-γ serum. A prolonged digestion of ECF₁ with trypsin removed intact γ from ECF₁, but did not alter the sensitivity of the ATPase to inhibition by the anti-γ serum. Proteolytic fragments were isolated from the trypsinized enzyme. They gave an immunoprecipitation band with the anti-γ serum, but none of the other subunit antisera. The antiδ serum detached ECF₁ from everted membrane vesicles and completely blocked both the ATP- and respiration-dependent pyridine nucleotide transhydrogenase, an energylinked membrane function. The δ antiserum had no effect on the ATPase activity of the ECF₁. The e antiserum stimulated the ATPase activity of purified ECF₁ as shown previously (P. P. Laget and J. B. Smith, Arch. Biochem. Biophys.197, 83, 1979), but strongly inhibited the holoenzyme in membrane vesicles. The α antiserum completely blocked the ATP-driven transhydrogenase. The same antiserum maximally inhibited the respiratory chain-driven reaction by only 35%. These observations indicate that the antiserum selectively affected energy transduction mediated by the ATPase. The protonmotive force generated by substrate oxidation was probably not dissipated by the ? antiserum. Adsorbing the δ or ? antiserum with everted membrane vesicles selectively removed those antibodies that reacted with membrane-bound ATPase. The adsorbed sera still reacted strongly with purified ECF₁, and prevented it from restoring ATP-dependent proton translocation in ECF₁-depleted vesicles. Therefore, it appears that more of the δ and the ? subunit is exposed in the purified ECF₁ molecule than in the membrane-bound enzyme.     

Identification of the altered subunit in the inactive F1ATPase of an Escherichia coli uncA mutant.

ATPase activity was restored to the inactive coupling factor, F₁ATPase, of $\text{Escherichia coli}$ strain AN120 ($\text{uncA401}$) by reconstitution of the dissociated complex with an excess of wild-type α subunit. Large excesses of α gave the highest levels of activity. The other subunits which are required for the reconstitution of ATPase activity, β and γ, did not complement the mutant enzyme. These results indicate that the α polypeptide of the AN120 ATPase is defective.     

A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

Comparative studies on mitochondrial coupling factor B and FB.

Beef heart mitochondrial protein factor F_B [Higashiyama $\text{et}\text{al}$, Biochemistry $\text{14}$, 4117–4121 (1975)] was purified and its properties were compared with those of coupling factor B. Both proteins stimulated ATP-driven NAD⁺ reduction in ammonia and EDTA-treated (AE-) submitochondrial particles, but the extent of stimulation (maximum activity of particles) was very low with F_B. F_B was found to be ineffective in stimulating P_i-ATP exchange in either AE-particles or reconstituted oligomycin-sensitive ATPase vesicles. Furthermore, F_B failed to stimulate ATP-driven NAD⁺ reduction activity of AE-particles in the presence of saturating amounts of dithiothreitol (DTT). DTT alone stimulates the particle activity extensively as reported earlier. Rabbit antiserum to F_B did not show a precipitin band with purified Factor B, nor did the antibody inhibit Factor B stimulated activity of the AE-particles. The data suggest that F_B and Factor B are two different molecular species with different functions and fail to provide evidence that F_B is a coupling factor.     

Specificity of association of a Ca2+/Mg2+ ATPase with cholinergic synaptic vesicles from Torpedo electric organ.

Cholinergic synaptic vesicles from the electric organ of $\text{Torpedo}$$\text{californica}$ have been subjected to analytical scale separation techniques not utilized in the isolation procedure, and the ATPase activity of separated fractions determined. Most of the ATPase activity migrated with the vesicles. Sensitivity of the ATPase activity to 16 potential inhibitors also was determined. Most of the ATPase activity was inhibited by low concentrations of 4-chloro-7-nitrobenzo-oxadiazole (NBD-C1) and dicyclohexylcarbodiimide (DCCD), but not by a water soluble carbodiimide. The close association of the ATPase with the vesicles and the pattern of inhibition obtained provide further support for the authentic presence of a membrane bound $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase in the cholinergic synaptic vesicle.     

Chlorpromazine induces membrane potential depolarization and ATP loss,and inhibits microsomal ATPase in soybean (Glycine Max L.) roots

In intact soybean roots, chlorpromazine causes a depolarization of the membrane potential at low concentrations (as low as 30 μM, half-maximally at about 150 μM), and induces a marked decrease in ATP levels at higher concentrations (half-maximal at about 0.5 mM) over longer periods of time. In root microsomal suspensions, chlorpromazine inhibits an apparently specific ATPase activity component (half-maximally at about 0.3 mM). Chlorpromazine inhibits $\text{N,N′-}\text{dicyclohexylcarbodiimide}$-, diethylstilbesterol- and azide-inhibited ATPase activities. On linear sucrose gradients, chlorpromazine inhibition of ATPase activity occurs in two peaks, at 1.12 g/ml and 1.14–1.17 g/ml, which may represent a tonoplast and plasma membrane ATPase, respectively. Neither peak corresponds to the F₁ ATPase. It is unclear whether ATPase inhibition or ATP loss is the cause of the membrane potential depolarization. Clearly chlorpromazine has multiple effects which are probably unrelated to its calmodulin-inhibition activity.     

A study of the surface-active properties of the Mg2+-activated ATPase from cytoplasmic membranes of Streptococcus faecalis

The surface activity and enzymic properties of the factor F₁, the catalytic moiety of Streptococcus faecalis H⁺-ATPase, has been studied at the air-water and phospholipid-water interfaces. F₁ does not interact with the monolayer phospholipids, hence its adsorption on a biological membrane must be due mainly to its recognition of proteins of the hydrophobic complex. The dimensions of the F₁ molecule at the air-water interface have been estimated. In the presence of Mg²⁺, base area is $\text{S = 1.8 · 10}{}^4\text{A}\text{?}{}^2$, height $\text{h = 27}\text{A}\text{?}$. Bearing in mind the size of a globular subunit, it follows from the measurements that the major F₁ subunits should all lie in the same plane. The ATPase activity of F₁ at the interface is inversely proportional to the monolayer density. With low density monolayer, the specific ATPase activity is higher at the interface than in the bulk of the solution.Adsorption of F₁ at the interface shifts the isoelectric point of the protein, apparently due to changes in its conformation. The findings are discussed relative to the proton-active transport mechanism.     

Estimations of membrane potentials in Streptococcus faecalis by means of a fluorescent probe

Membrane potentials in $\text{Streptococcus faecalis (faecium)}$ were estimated by means of the fluorescent probe, 1,1′-dihexyl-2,2′-oxycarbocyanine. In the absence of D-glucose the potential was ?60 to ?70 mV for normal cells suspended in 0.09 M NaCl + 0.01 M Tris-HCl at pH 7.5. When metabolism was initiated by the addition of D-glucose the cells became hyperpolarized (internal becomes more negative). The new potential, ?130 to ?140 mV, was fully manifested 35 seconds after the glucose was added. N,N′-dicyclohexylcarbodiimide, a membrane ATPase inhibitor prevented the hyperpolarization seen upon the addition glucose. The results are consistent with the view that glycolyzing cells generate a considerasble electrical potential across the cell membrane.     

Restoration of coupling factor activity to Escherichia coli ATPase missing the delta subunit.

We separated the two minor subunits (δ and ε) of the $\text{E. coli}$ ATPase from the major subunits (α, β, and γ). The minor subunit fraction was obtained by treating purified ATPase with pyridine following the procedure that Nelson $\text{et al}$. (J. Biol. Chem. 348, 2049 [1973]) used to separate the subunits of chloroplast ATPase. The minor subunit fraction restored the capacity of ATPase lacking the delta subunit to recombine with ATPase-depleted membrane vesicles and to reconstitute energy coupling to the transhydrogenase and oxidative phosphorylation in the vesicles. These results clearly implicate the delta subunit in the attachment of the ATPase to the membrane.     

Interaction of Mg+2 with beef heart mitochondrial ATPase (F1).

The soluble mitochondrial ATPase, F₁, can be slowly inactivated by incubation with Mg⁺² in a manner consistent with the observations of Moyle and Mitchell $\text{(}\text{FEBS}\text{Lett}\text{.}\text{56}$, 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F₁ which has been incubated with Mg⁺². This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F₁ concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F₁ is unstable if incubated with EDTA in the absence of free nucleotides or Mg⁺². The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg⁺² in excess of the EDTA or 50 μM ADP stabilize the F₁ against the inactivation but cannot reverse prior denaturation.     

Nucleotide sequence of the genes for β and ε subunits of proton-translocating ATPase from Escherichia coli

The 1855-nucleotide long DNA sequence of part of the gene cluster for the proton-translocating ATPase from $\text{E. coli}$ was determined by the method of Maxam-Gilbert. The sequence covers the genes for the β and ε subunits of F₁ along with the flanking region. The amino acid sequence of these subunits deduced from the nucleotide sequence indicates that the β and ε subunits have 459 and 138 amino acids, respectively. The possible secondary structure of the both subunits was estimated from the deduced primary structures. A possible nucleotide binding site in the β subunit is also discussed on the basis of the primary and secondary structures. The codons used in the genes for all the components of F₁F₀ were different in different genes, suggesting that the amount of each subunit in the F₁F₀ is determined to some extent on a translational level.     

Electron-Microscopic Demonstration of a “Head and Stalk” Structure of the Leaf Vacuolar ATPase in Mesembryanthemum crystallinum L.

The structure of the vacuolar ATPase from mesophyll tonoplasts of Mesembryanthemum crystallinum has been studied by electron microscopy using negatively stained specimens of membrane-bound and detergent-solubilized ATPase molecules. We observed a high density of particles on the surface of tonoplast vesicles and “head and stalk” structures on the edge of the membrane, similar to the F₀F₁-ATPases of mitochondrial and chloroplast membranes. The staining conditions, which are often critical for such small objects, were improved by using methylamine tungstate as negative stain for the membrane-bound ATPase. Compared to other staining solutions generally applied, dissociation of the F₁-like enzyme complex from the membrane was best prevented and structural damage of the vesicles was least observed with methylamine tungstate. In freeze-fracture electron microscopy of tonoplast vesicles, where dissociation never occurs since no detergent is used, we also observed “head and stalk” structures on the edge of the membranes, beside many particles on the fracture faces. The detergent-solubilized ATPase forms string-like structures, caused by the aggregation of the hydrophobic membrane-embedded F₀-like part of the enzyme. After negative staining the F₁-like enzyme complex is arranged alternately along both sides of the string and connected by a narrow stalk.     

Mg2+-induced ADP-dependent inhibition of the ATPase activity of beef heart mitochondrial coupling factor F1.

Incubation of F₁ in the presence of Mg²⁺ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg²⁺ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F₁ treated to remove tightly bound nucleotides was preincubated in the presence of Mg²⁺. Mg²⁺-induced inhibition of ATPase activity of nucleotide-depleted F₁ can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg²⁺-induced inhibition of the ATPase activity of F₁ is due to the formation of an inactive F₁. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, $\text{86}$, 434/ is directed to F₁ component of the complete mitochondrial ATPase system.     

Role of the plasma membrane in the mechanism of cholesterol efflux from cells

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [³H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu₅AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for plasma membrane vesicles of Fu₅AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu₅AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu₅AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for cholesterol efflux from these cell lines.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with $\text{ascorbate}\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylenediamine}$. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low .     

Evidence for an electrogenic ATPase in microsomal vesicles from pea internodes

The transmembrane electropotential of microsomal vesicles from pea internode segments, monitored by equilibrium distribution of the permeant anion SCN^?, is strongly hyperpolarized when ATP is present in the incubation medium.The stimulation of SCN^? uptake by ATP is rather specific with respect to the other nucleoside di- and triphosphates tested: ADP, GTP, CTP and UTP. ATP-stimulated SCN^? uptake is strongly inhibited by ATPase inhibitors such as $\text{p-}\text{chloromercuribenzenesulphonate}$ and $\text{N,N}{}^{\mathrm{\prime }}\text{-}\text{dicyclohexylcarbodiimide}$ and by 2.5% toluene/ethanol (1 : 4, v/v), the latter being a treatment which makes the vesicles permeable. On the contrary, oligomycin is almost ineffective in influencing ATP-induced SCN^? uptake. The proton conductor carbonyl cyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ strongly inhibits ATP-stimulated SCN^? uptake. The effect of ATP on SCN^? uptake depends on the pH of the medium, the maximum being reached at about pH 7.0.These data support the view that microsomal fractions from pea internodes contain membrane vesicles endowed with a membrane-bound ATPase coupling ATP hydrolysis to electrogenic transport of ions, probably H⁺.