Solubilization of a phospholipid-stimulated adenosine triphosphatase complex from membranes of Escherichia coli.

A phospholipid-stimulated adenosine triphosphatase (ATPase) complex was solubilized and partially purified from membrane particles of Escherichia coli ML308-225. The complex was of large molecular size and contained 16 polypeptides, five of which were subunits of the F₁-type ATPase of E. coli. Components of the respiratory chain were absent. Enzyme activity was stimulated by lysophosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, and cardiolipin but not by phosphatidylethanolamine. The ATPase activity of the complex was inhibited by N,N′-dicyclohexylcarbodiimide and by Dio-9 at lower inhibitor:protein ratios than required for inhibition of the F₁-type ATPase of E. coli. However, the ATPase complex was less sensitive than the membrane-bound enzyme to inhibition by these compounds.     

Purification and characterization of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase complex from membranes of Escherichia coli.

The energy transducing adenosine 5′-triphosphatase (ATPase) complex was extracted with deoxycholate from $\text{Escherichia coli}$ membranes and purified 20–25 fold. The detergent-solubilized ATPase complex was inhibited more than 80% by dicyclohexylcarbodiimide (DCCD). Its sedimentation velocity coefficient was 14.7s in the presence of deoxycholate. Phospholipid stimulated its hydrolytic activity and maximized DCCD sensitivity. These parameters clearly differentiate the ATPase complex from the DCCD-insensitive, soluble ATPase prepared by extraction with EDTA at low ionic strength. The purified ATPase complex showed twelve discrete bands on lauryl sulfate gel electrophoresis. Five of these components co-electrophresed with subunits of soluble ATPase. Of the seven additional components, primarily two were precipitated with antibody to soluble ATPase. The protein which specifically reacts with DCCD co-migrated with one of these subunits.     

Size and molecular parameters of adenosine triphosphatase from Escherichia coli.

The Mg2+- and Ca2+-stimulated ATPase (bacterial coupling factor) has been investigated in solution with different independent techniques. The molecular weight of the five-subunit enzyme was found to be 345,000 +/- 5,000 by means of light scattering, 350,000 by sedimentation equilibrium experiments, and 358,000 by means of small-angle x-ray scattering. The radius of gyration was found to be 41.9 A, the volume 7.39 x 10(5) A3, and the surface to volume ratio 5.5 x 10(-2) A-1 from small-angle x-ray scattering measurements of the enzyme in solution. The degree of hydration was found to be 0.62 ml of H2O/g of ATPase. The translational diffusion coefficient was determined to be 3.47 x 10(-7) cm2 s-1 by means of inelastic light scattering. The distribution of the scattered intensity near the origin appears to be bimodal, suggesting that the ATPase molecule is composed of spherical parts bound together by a flexible polypeptide chain. The largest dimension of the ATPase in solution is 120.0 A, determined from the pair distribution function.     

Non-covalently bound adenine nucleotides in adenosine triphosphatase of Escherichia coli.

The term, xeroderma pigmentosum variants designates patients who suffer from the clinical manifestations of the disease, but whose cells have normal rates of excision repair of UV-induced lesions in DNA. In contrast to normal human fibroblasts, if cells from such variants are maintained in medium containing caffeine from immediately following exposure to UV until the survivors have undergone three doublings, the cytotoxic and mutagenic effect of UV light is dramatically increased. In the presence of 0.7mM caffeine, the slope of the UV survival curve increases $\text{ca.}$ 3-fold. Similarly, the slope of the curve describing the frequency of mutations to azaguanine resistance induced by UV as a function of dose is $\text{ca.}$ 3-fold steeper.     

Inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by dicyclohexylcarbodi-imide.

The inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by DCCD (dicyclohexylcarbodi-imide) is studied under conditions of varying KCl concentration. An increase in K+ concentration and in other cations causes an increase in the DCCD sensitivity of the enzyme, as well as significant changes in the kinetic parameters.     

Isolation of Escherichia coli mutants with an adenosine triphosphatase insensitive to aurovertin.

Energy-transducing adenosine triphosphatase (ATPase) from Escherichia coli is inhibited by aurovertin. Aurovertin-resistant mutants were generated by nitrosoguanidine mutagenesis of E. coli AN180, whose growth on a nonfermentable carbon source was blocked by aurovertin. The ATPase activity of cell extracts from 15 different mutants (designated MA1, MA2, MA3, etc.) was found to be at least 20 times less sensitive to aurovertin than that from the parent strain. The aurovertin-resistant mutants did not show cross-resistance towards a number of ATPase inhibitors including azide, dicyclohexylcarbodiimide, quercetin, 7-chloro-4-nitrobenzofurazan, and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Aurovertin inhibited the energization brought about by addition of ATP to E. coli AN180 membrane vesicles; it was without effect on MA1 and MA2 membrane vesicles energized by ATP. The mutation in MA1, like other mutations of the ATPase complex, maps in the unc region of the bacterial chromosome.     

Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from Escherichia coli.

The portion of Escherichia coli adenosine triphosphatase (ATPase) which is peripheral to the membrane (ECFl) is composed of five separate polypeptides referred to as alpha, beta, gamma, delta, and epsilon. Treating purified ECFl with pyridine precipitated the three larger polypeptides (alpha, beta, and gamma), but the two smaller ones (delta and epsilon), which represent only about 10% of ECFl, remained in solution. After removing the pyridine, both delta and epsilon were active and both were obtained in essentially pure form after chromatography on a single molecular-seive column. epsilon strongly inhibited the ATPase activity of ECFl, indicating that epsilon has a regulatory role in the enzyme. epsilon inhibited ECFl missing delta, indicating that delta is not required for inhibition by epsilon. However, enzyme containing just the alpha and beta subunits, which was prepared by treating ECFl with a protease, was fully active hydrolytically but not at all sensitive to inhibition by epsilon. This result suggests that the gamma polypeptide is required for the inhibition of the ATPase by epsilon. delta restored the capacity of ECFl missing delta to recombine with ECFl-depleted membrane vesicles. The ECFl, which became attached to the vesicles by the added delta, was functional in energy transduction, as evidenced by the coupling of ATP hydrolysis to the transhydrogenase reaction in the vesicles. The rebinding of ECFl missing delta was directly proportional to the amount of delta added until all the ECFl receptors in the membranes were occupied. delta may be a stalk which connects the Fl headpiece to the membrane, since the attachment of ECFl to the membrane exhibited an absolute dependence on delta. Although delta is known to have an apparent molecular weight of about 20,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, the active delta eluted from a molecular-seive column with an apparent molecular weight of about 35,000, suggesting that in the active form delta is a dimer or rather elongated in shape. The active epsilon subunit eluted from the same column with an apparent molecular weight of about 16,000.     

Structure-function relationships of domains of the delta subunit in Escherichia coli adenosine triphosphatase.

The topology of the and subunit of the Escherichia coli adenosinetriphosphatase (ECF1) has been explored by proteinase digestion and chemical labeling methods. The delta subunit of ECF1 could be cleaved selectively by reaction of the enzyme complex with very low amounts of trypsin (1:5000, w/w). Cleavage of the delta subunit occurred serially from the C-terminus. The N-terminal fragments of the delta subunit remained bound to the core ECF1 complex through sucrose gradient centrifugation, indicating that part of the binding of this subunit involves the N-terminal segment. ECF1, in which around 20 amino acids had been removed from the C-terminus of delta, still bound to ECF0 but DCCD sensitivity of the ATPase activity was lost. When ECF1 was reacted with N-ethyl[14C]maleimide ([14C]NEM) in the native state, only one of the two Cys residues on the delta subunit was modified. This residue, Cys-140, was also labeled in ECF1F0. Cys-140 was shown to be involved in the disulfide bridge between alpha and delta subunits that is generated when ECF1 is treated with CuCl2. Thus, the C-terminal part of the delta subunit around Cys-140 can interact with the core ECF1 complex. These results suggest a model for the delta subunit in which the central part of polypeptide is a part of the stalk, with both N- and C-termini associated with ECF1.     

Interaction of Escherichia coli adenosine triphosphatase with aurovertin and citreoviridin: inhibition and fluorescence studies.

Aurovertins B and D inhibited the adenosine triphosphatase (ATPase) activity of soluble Escherichia coli coupling factor ATPase (BF1) isolated from wile-type E. coli K-12. Half inhibition was obtained with 2 microns aurovertin B and 0.9 microns aurovertin D. Aurovertins B and D had no inhibitory effect on BF1 isolated from the aurovertin-resistant E. coli mutant MA12. Acetylation or saponification of aurovertin D yielded a derivative which was devoid of inhibitory effect on BF1. Citreoviridin also inhibited wild-type BF1 but with much less efficiency (half inhibition at 60 microns) than aurovertin. Citreoviridin had no effect on the aurovertin-resistant BF1. The fluorescence intensity of aurovertins B and D was markedly enhanced upon addition to purified BF1. There was no enhancement of fluorescence when the aurovertins were added to BF1 isolated from the aurovertin-resistant mutant. The fluorescence of the aurovertin-BF1 complex was enhanced by adenosine 5'-diphosphate and by low concentrations of adenosine 5'-triphosphate. The adenosine 5'-diphosphate-enhanced fluorescence of the aurovertin-BF1 complex was quenched by high concentrations of adenosine 5'-triphosphate or by MG2+. Aurovertin bound selectively to the beta subunit of BF1 isolated from wile-type cells. By complementation assays in vitro, using a reconstituted system made of subunits isolated from wild-type and aurovertin-resistant BF1, it was shown that the altered peptide in aurovertin-resistant BF1 was the beta subunit.     

A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli. I. Copurification and separation.

Adenosinetriphosphatase (ATPase) [EC 3.6.1,3] activity has been found to exist in most preparations of DNA-dependent RNA polymerase [EC 2.7.7.6] obtained from Escherichia coli by a number of purification procedures so far established. Electrophoretic analysis on polyacrylamide gels demonstrated that ATP hydrolysis and RNA synthesis were catalyzed by two distinct enzyme proteins. It appears that the two enzymes are associated or have similar molecular properties. Separation of the two enzymes, the object of the present work, was achieved by three independent methods: ion exchange chromatography on a phosphocellulose column, electrophoresis in glycerol gradients, or high-salt glycerol gradient centrifugation.     

Solubilization of a functionally active proline carrier from membranes of Escherichia coli with an organic solvent.

A proline transport carrier was extracted from the membranes of Escherichia coli with acidic n-butanol. Vesicles reconstituted from the butanol extract and E. coli phospholipids and preloaded with K⁺ showed rapid uphill uptake of proline when energy was supplied as a membrane potential introduced by K⁺-diffusion via valinomycin. Proline uptake by the reconstituted vesicles, like that of intact cells and isolated membrane vesicles, was inhibited by 3,4-dehydroproline, SH reagents, and a proton conducting uncoupler. Reconstituted vesicles of mutants defective in proline transport showed little or no proline uptake. The proline carrier was partially purified from the extract and separated from the bulk of phospholipids on Sephadex LH-20.     

Isolation and properties of anion-sensitive adenosine triphosphatase from erythrocyte membranes

Using the non-ionic detergent Triton X-100 and gel-chromatography, an anion-sensitive ATPase was isolated from rat and rabbit erythrocyte membranes. The ATPase preparations possess no Na, K- or Mg, Ca-ATPase activities. ATPase from rat erythrocyte membranes is made up of five subunits with molecular weights of 58 000 (alpha), 50 000 (beta), 36 000 (gamma), 25 000 (delta) and 12 000 (epsilon) and can be represented by the formula alpha 3 beta 3 gamma delta epsilon.     

Characterization of the inhibitory (epsilon) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli

The inhibitory subunit (epsilon) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12 (lambda) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active epsilon from both strains was found to be a globular protein with a Stokes' radius of 18--19 A. Circular dichroism spectra suggested an alpha-helix content of approximately 40%. The molecular weight of epsilon was approximately 15000--16000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20,w of epsilon was approximately 1.6 s-1. Inhibition of the purified F1 ATPase by epsilon displayed noncompetitive kinetics with a Ki of approximately 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme--epsilon mixture. [125I]epsilon which was incorporated into ECF1 was readily displaced by unlabeled epsilon. epsilon had no significant effect on the ATPase activity of "native" or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the epsilon-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that epsilon inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.