Membrane-bound ATPase Research Articles

Biochemical Studies on the Protective Effects of Picrorhiza kurroa in Experimentally Induced Hepatitis in Rats.

The effect of pre-treatment with an alcoholic extract of Picrorhiza kurroa, a herbal drug, on D-galactosamine-induced hepatitis in rats was investigated with respect to the activities of serum marker enzymes, glycolytic enzymes, gluconeogenic enzymes, glucose-6-phosphate dehydrogenase, and membrane-bound ATPases and the levels of lipid peroxides and hepatic thiols. Prior oral treatment with P. kurroa extract significantly prevented the D-galactosamine-induced increases in the activities of serum marker enzymes, glycolytic enzymes, glycogen phosphorylase, and glucose-6-phosphate dehydrogenase and the decreases in the activities of key gluconeogenic enzymes and membrane-bound ATPases in the liver. The extract also exerted an antioxidant effect against D-galactosamine-induced hepatitis by blocking the induction of lipid peroxidation and by preventing the depletion of hepatic thiols. The present findings confirm that P. kurroa is hepatoprotective in experimentally induced hepatitis in rats.

Vitamin E and ATPases: protection of ATPase activities by vitamin E supplementation in various tissues of hypercholesterolemic rats.

It has been shown that the lipid composition of plasma membrane can be modified in vivo by dietary fat. It has also been observed that an increase in the cholesterol content of plasma membranes results in decreased activities of ATPases. In the present study, we evaluated the changes in the activities of ATPases from erythrocytes, hepatocytes, and kidney cortex caused by cholesterol-rich diet in rats and subsequently examined the role of vitamin E administration on the cholesterol-induced effects in these tissues. Administration of hypercholesterolemic diet to the rats for 4.5 months, significantly decreased membrane Na(+)-K(+)-ATPase and Ca+2-ATPase activities in comparison to the controls in all tissues studied. Vitamin E supplementation to the hypercholesterolemic rats led to a recovery in membrane ATPase activities. In conclusion, vitamin E supplementation to the rats provided protection against hypercholesterolemic diet-induced impairment of membrane-bound ATPases.

Ketanserin inhibits digoxin-induced arrhythmias in the anaesthetized guinea-pig.

Digoxin inhibits the membrane-bound ATPase enzyme, resulting in a rise in intracellular sodium and activated outward potassium current, predisposing to arrhythmias. In this study, the effect of ketanserin, thought to block outward potassium currents, was investigated on digoxin-induced arrhythmias. Twenty-four guinea-pigs were studied in four groups (control, ketanserin 0.5 mg/kg, ketanserin 1 mg kg, ketanserin 2 mg/kg). Under pentobarbital anaesthesia (40 mg/kg), 15 min after injection of saline or ketanserin, digoxin (0.6 mg/kg) was administered through the jugular vein. Carotid artery blood pressure and electrocardiogram (ECG) were recorded. The time for the onset of the first arrhythmia and incidence of ventricular tachycardia (VT), ventricular fibrillation (VF), and premature ventricular contraction (PVC) were determined. Arrhythmias were scored according to the MacLeod scale. Ketanserin produced minor haemodynamic effects and lacked, by itself, arrhythmogenic effects at the doses studied. However, it increased the time for the onset of the first digoxin-induced arrhythmia and decreased the incidence of VT, VF and PVC. We conclude that ketanserin inhibits digoxin-induced arrhythmias in guinea-pigs.

The Proteolipid of the A1A0ATP Synthase from Methanococcus jannaschii Has Six Predicted Transmembrane Helices but Only Two Proton-translocating Carboxyl Groups

The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F(1)F(0) ATPases and most archaeal A(1)A(0) ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H(+) ATPases and has important mechanistic consequences for the structure and function of H(+) ATPases in general.

His15 of Subunit a of the Escherichia coli ATP Synthase Is Important for the Structure or Assembly of the Membrane Sector Fo

Approximately 37 amino acids at the amino-terminus of subunit a of the Escherichia coli ATP synthase are found localized to the periplasm. Results indicate that a single amino acid substitution, H15D, disrupts assembly of subunit a and causes a loss of ATP synthase function. In this study, a conserved region of nine amino acids, 11–19, was initially mutagenized randomly, generating no mutants that could grow on succinate-minimal medium. Subsequent mutagenesis, confined to residues His14, His15, and Asn17, indicated that constructs containing H15D were the most deleterious. Four single mutants were constructed and analyzed: H15A, H14D, H15A, and H15D. Only H15D was significantly impaired, with respect to ATP-driven proton translocation, passive proton permeability through Fo, and sensitivity of membrane-bound ATPase to DCCD. Immunoblot analysis indicated very low levels of subunit a from H15D. Cysteine mutations were constructed at positions 14, 15, 17, and 18. Residues 14, 15, and 17 were shown to be accessible in the periplasmic space, while residue 18 was not, indicating that this region was stably folded. While both His14 and His15 are conserved among a group of bacteria, results presented here indicate that they are not equivalent, and that a specific role for His15 in the assembly or structure of the ATP synthase is supported.

Presence of Na+-stimulated P-type ATPase in the membrane of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum.

It was found that a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, possesses a membrane-bound ATPase, which was activated specifically by Na+. The Na+-stimulated ATPase activity reached a maximum value at 200 mM NaCl. In the presence of 200 mM NaCl, the activity was drastically reduced by vanadate, a potent inhibitor of P-type ATPase, with a half-maximal inhibition at 1 microM. Incubation of the membranes with [gamma-32P]ATP followed by acidic lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the existence of two phosphorylated intermediates with apparent molecular masses of 60 and 100 kDa. Only phosphorylation of the 100-kDa polypeptide was inhibited by vanadate. The membrane extract containing Na+-stimulated ATPase, when reconstituted into soybean phospholipid vesicles, exhibited 22Na+ transport by the addition of ATP, which was inhibited by vanadate and gramicidin. It is likely that the Na+-stimulated ATPase belongs to P-type and is involved in Na+ transport.

DL-.ALPHA.-Lipoic Acid Supplementation Attenuates the Age-Related Changes in Membrane-Bound ATPases in Rats.

A great deal of attention has been focused on the possible therapeutic implications of lipoic acid as a potent antioxidant. Lipoic acid serves as a vital cofactor for α-keto-acid dehydrogenase and is well known for its free radical-quenching effect. We evaluated the age-associated alterations in membrane-bound enzymes, glutathione and lipid peroxidation in young and aged rats in response to lipoate supplementation. In aged rats, the level of glutathione and the activities of membranebound enzymes were lower, whereas thiobarbituric acid reactive substances (a marker of free radical damage) were higher than those in the young rats. Intraperitoneal administration of lipoic acid to the aged rats led to a time-dependent reduction in lipid peroxidation and elevation in the activities of membrane-bound enzymes as well as in the level of glutathione. Our observations highlight the protective effect of lipoic acid in age-associated free radical-induced oxidative stress, thus proving its efficacy as a potent antioxidant.

Activation and deactivation of F0F1-ATPase in yeast mitochondria.

The regulation of membrane-bound proton F0F1 ATPase by the protonmotive force and nucleotides was studied in yeast mitochondria. Activation occurred in whole mitochondria and the ATPase activity was measured just after disrupting the membranes with Triton X-100. Deactivation occurred either in whole mitochondria uncoupled with FCCP, or in disrupted membranes. No effect of Triton X-100 on the ATPase was observed, except a slow reactivation observed only in the absence of MgADP. Both AMPPNP and ATP increased the ATPase deactivation rate, thus indicating that occupancy of nucleotidic sites by ATP is more decisive than catalytic turnover for this process. ADP was found to stimulate the energy-dependent ATPase activation. ATPase deactivated at the same rate in uncoupled and disrupted mitochondria This suggests that deactivation is not controlled by rebinding of some soluble factor, like IF1, but rather by the conversion of the F1.IF1 complex into an inactive form.

The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes.

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 microM) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. Ouabain (Na+/K(+)-ATPase inhibitor) had no effect. Bafilomycin A1 (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 microM, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.

Deletion of transmembrane domain 12 of CDR1, a multidrug transporter from Candida albicans, leads to altered drug specificity: expression of a yeast multidrug transporter in baculovirus expression system.

Cdr1p, an ATP-binding cassette transporter from the pathogenic yeast Candida albicans, confers resistance to several unrelated drugs including anti-Candida drugs (Prasad et al., 1995b). We demonstrate that the deletion of 237 bp (79 aa) from the 3' end of CDR1 (which encompasses the transmembrane domain (TM) 12 of the putative transporter) did not result in the total loss of its ability to efflux cytotoxic agents. While the expression of deltaCDR1 in yeast resulted in impaired sensitivity to drugs like cycloheximide, anisomycin, sulfomethuron methyl and antifungal nystatin, its ability to confer resistance remained unaltered to drugs such as o-phenanthroline, 4-nitroquinoline-N-oxide, cerulenin, azoles, oligomycin, erythromycin, and benomyl. Similar to human MDR1p. Cdr1p might also have localized drug binding sites in TM 12, but that might not be the case for all the drugs. The TM 12 deletion also did not lead to any significant impairment in NTPase activities. Both ATPase and UTPase activities of complete Cdr1p and deltaCdr1p were not significantly altered, as was the case with respect to their ability to efflux Rh123 and steroid hormone like [3H]-beta-estradiol. To further dissect the functionality of Cdr1p, its truncated version was overexpressed in a baculovirus-insect cell expression system. The synthesis of deltaCdr1p in Sf9 cells was temporally regulated as a function of the baculovirus polyhedrin gene promoter. The Sf9 derived deltaCdr1p was approximately 130 kDa, which was lower than the expected size, probably due to the differences in glycosylation. This, however, did not affect the functionality of deltaCdr1p. The deletion of TM 12 did not affect the targeting of the protein and deltaCdr1p was exclusively localized in plasma membrane of Sf9 cells as detected by immunofluorescence. The expression of deltaCdr1p in the baculovirus-insect expression system generated a high drug-stimulated plasma membrane-bound ATPase activity which was not demonstrable when deltaCdr1p was expressed in yeast.

Effects of High Pressure on Inactivation Kinetics and Events Related to Proton Efflux in Lactobacillus plantarum.

Knowledge of the mechanism of pressure-induced inactivation of microorganisms could be helpful in defining an effective, relatively mild pressure treatment as a means of decontamination, especially in combination with other physical treatments or antimicrobial agents. We have studied the effect of high pressure on Lactobacillus plantarum grown at pH 5.0 and 7.0. The classical inactivation kinetics were compared with a number of events related to the acid-base physiology of the cell, i.e., activity of F(0)F(1) ATPase, intracellular pH, acid efflux, and intracellular ATP pool. Cells grown at pH 5.0 were more resistant to pressures of 250 MPa than were cells grown at pH 7.0. This difference in resistance may be explained by a higher F(0)F(1) ATPase activity, better ability to maintain a DeltapH, or a higher acid efflux of the cells grown at pH 5.0. After pressure treatment at 250 MPa, the F(0)F(1) ATPase activity was decreased, the ability to maintain a DeltapH was reduced, and the acid efflux was impaired. The ATP pool increased initially after mild pressure treatment and finally decreased after prolonged treatment. The observations on acid efflux and the ATP pool suggest that the glycolysis is affected by high pressure later than is the F(0)F(1) ATPase activity. Although functions related to the membrane-bound ATPase activity were impaired, no morphological changes of the membrane could be observed.

Preparation and characterization of everted membrane vesicles from cells of Staphylococcus aureus.

We developed a method for the preparation of everted membrane vesicles from cells of Staphylococcus aureus. The cells were first treated with ampicillin to weaken the peptidoglycan layer, then the cells were passed through a French press cell. The resulting vesicles were roughly 0.1 microm in diameter, judging from electron microscopic observations. We detected fairly high membrane-bound ATPase activity in the membrane vesicles. We observed respiratory-driven quenching of quinacrine fluorescence, which indicates that inward H+ transport took place. These results indicate that the vesicles are everted. We characterized the membrane-bound ATPase. We also detected Na+/H+ antiport, erythromycin/H+ antiport and chloramphenicol/H+ antiport activities in the membranes of S. aureus.

Characterization of a mutant of Lactococcus lactis with reduced membrane-bound ATPase activity under acidic conditions.

A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0 while the level of increase in the mutant was only 1.6. Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1 beta subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F1 beta subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.

Near-UV-induced absorbance change and photochemical decomposition of ergosterol in the plasma membrane of the yeast Saccharomyces cerevisiae.

When cells of the yeast Saccharomyces cerevisiae were exposed to near-UV (300-400 nm), their absorption spectra changed slightly within the range 220-300 nm with increasing dosage. Difference spectra, calculated by substracting the curve recorded in cells exposed to near-UV from the curve of unexposed cells, decreased with increasing dosage over a broad band with peaks at 272, 282 and 295 nm and a shoulder at 265 nm. These peaks were in agreement with the absorption maxima of ergosterol, which is one of the major components of the plasma membrane of yeast. Near-UV radiation induced a simultaneous decrease in absorption spectra and reduction of ergosterol content in the plasma membrane. Photochemical decomposition of ergosterol by near-UV radiation was revealed in vivo, although ergosterol is generally known to be photoconverted to previtamin D2 industrially by UV radiation in vitro. In order to remove photosensitizers, liposomes were prepared from phospholipids and glycolipids, with or without ergosterol from purified yeast plasma membranes. Liposomal ergosterol in the orientated state was photochemically decomposed by near-UV radiation but ergosterol in the disorientated state in a homogeneous solution was not. Near-UV radiation also induced a decrease in activity of membrane-bound ATPase. Dose-response curves for the reduction of ATPase activity were similar to that for decomposition of ergosterol, suggesting that near-UV caused membrane function damage.

An Na+-pumping V1V0-ATPase complex in the thermophilic bacterium Clostridium fervidus.

Energy transduction in the anaerobic, thermophilic bacterium Clostridium fervidus relies exclusively on Na+ as the coupling ion. The Na+ ion gradient across the membrane is generated by a membrane-bound ATPase (G. Speelmans, B. Poolman, T. Abee, and W. N. Konings, J. Bacteriol. 176:5160-5162, 1994). The Na+-ATPase complex was purified to homogeneity. It migrates as a single band in native polyacrylamide gel electrophoresis and catalyzes Na+-stimulated ATPase activity. Denaturing gel electrophoresis showed that the complex consists of at least six different polypeptides with apparent molecular sizes of 66, 61, 51, 37, 26, and 17 kDa. The N-terminal sequences of the 66- and 51-kDa subunits were found to be significantly homologous to subunits A and B, respectively, of the Na+-translocating V-type ATPase of Enterococcus hirae. The purified V1V0 protein complex was reconstituted in a mixture of Escherichia coli phosphatidylethanolamine and egg yolk phosphatidylcholine and shown to catalyze the uptake of Na+ ions upon hydrolysis of ATP. Na+ transport was completely abolished by monensin, whereas valinomycin stimulated the uptake rate. This is indicative of electrogenic sodium transport. The presence of the protonophore SF6847 had no significant effect on the uptake, indicating that Na+ translocation is a primary event and in the cell is not accomplished by an H+-translocating pump in combination with an Na+-H+ antiporter.

Molecular bases of three characteristic phenotypes of pneumococcus: optochin-sensitivity, coumarin-sensitivity, and quinolone-resistance.

Streptococcus pneumoniae is uniquely sensitive to amino alcohol antimalarials in the erythro configuration, such as optochin, quinine, and quinidine. The protein responsible for the optochin (quinine)-sensitive (Opts, Qins) phenotype of pneumococcus is the proteolipid c subunit of the FzeroF1 H(+)-ATPase. OptR/QinR isolates arose by point mutations in the atpC gene and produce different amino acid changes in one of the two transmembrane alpha-helices of the c subunit. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opts) and M222 (an OptR strain produced by interspecies recombination between pneumococcus and S. oralis), and S. oralis (OptR) revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin, quinine, and related compounds specifically inhibited the membrane-bound ATPase activity. Equivalent differences between Opts/Qins and OptR/QinR strains, both in growth inhibition and in membrane ATPase resistance, were found. Pneumococci also show a characteristic sensitivity to coumarin drugs, and a relatively high level of resistance to most quinolones. We have cloned and sequenced the gyrB gene, and characterized novobiocin resistant mutants. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates. This residue position is equivalent to Val-120 of Escherichia coli ryGB, a residue that lies inside the ATP-binding domain but is not involved in novobiocin binding in E. coli, as revealed by crystallographic data. In addition, the genes encoding the ParC and ParE subunits of topoisomerase IV, together with the region encoding amino acids 46 to 172 (residue numbers as in E. coli) of the pneumococcal ryGA subunit, were characterized in respect to fluoroquinolone resistance. The gyrA gene maps to a physical location distant from the gyrB and parEC loci on the chromosome. Ciprofloxacin-resistant (CpR) clinical isolates had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level CpR), or in both resistance-determining regions of ParC and GyrA (high-level CpR). Mutations were found in residue positions equivalent to Ser-83 and Asp-87 of the E. coli GyrA subunit. Transformation experiments demonstrated that topoisomerase IV is the primary target of ciprofloxacin, DNA gyrase being a secondary one.

Subunit a of proton ATPase F 0 sector is a substrate of the FtsH protease in Escherichia coli

Escherichia coli FtsH is a membrane-bound ATPase with a proteolytic activity against the SecY subunit of protein translocase. We now report that subunit a of the membrane-embedded F 0 part of H +-ATPase is another substrate of FtsH. Pulse-chase experiments showed that subunit a is unstable when it alone (without F 0 subunits b and c) was oversynthesized and that it is stabilized in the ftsH mutants. Selective and ATP-dependent degradation of subunit a by purified FtsH protein was demonstrated in vitro. These results suggest that FtsH serves as a quality-control mechanism to avoid potentially harmful accumulation of free subunit a in the membrane.

FtsH (HflB) Is an ATP-dependent Protease Selectively Acting on SecY and Some Other Membrane Proteins

The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins. SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein). We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH. We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini. Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (A1) is essential for ATPase activity and in vivo functioning of FtsH. Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro. Mutant proteins without ATPase activity were inactive in proteolysis. A zinc binding motif mutant showed a decreased proteolytic activity. SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation. These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity. FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation.

A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY.

Escherichia coli FtsH (HflB), a membrane-bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit. We have now isolated SecY-stabilizing mutations that cause an amino acid substitution in the HflK-HflC membrane protein complex. Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK-hflC did not stabilize SecY. Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY. These results raise the possibility that even the wild-type HflKC protein acts to antagonize FtsH. Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein. Furthermore cross-linking, co-immunoprecipitation, histidine-tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro. Finally, purified HflKC protein inhibited the SecY-degrading activity of purified FtsH protein in vitro. These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC.

High Expression of Plasmid-Encoded Tetracycline Resistance Gene inE. coliCauses a Decrease in Membrane-Bound ATPase Activity

Expression of the plasmid pBR322-encoded tetracycline-resistant gene (tet) is known to cause other pleiotropic effects in addition to mediating the efflux of tetracycline from bacterial host cells. We have recently reported that expression of thetetgene in plasmid pKH47, a high-copy-number derivative of pBR322, causes growth inhibition ofEscherichia colicells harboring this plasmid. In this paper we report that reduced membrane-bound ATPase activity is found inE. colicells containing plasmid pKH47. This effect is dependent on the presence of an intacttetgene and reduces the ability of the cells to grow in a minimum medium containing succinate as the sole carbon source. The same effect is more dramatically observed in cells containing an unrelated plasmid in whichtetgene expression is under the control of thetacpromoter.