Purification and characterization of an ATPase from human liver which catalyzes ATP hydrolysis in the presence of the conjugates of bilirubin bile acids and glutathione

An ATPase has been purified from the membrane fraction of human liver which catalyzes ATP in the presence of bilirubin ditaurate, lithocholic acid 3-O-sulfate and lithocholic acid 3-O-glucuronide as well as dinitrophenylglutathione and other glutathione conjugates. Its subunit Mr value (38,000) and immunological properties are similar to dinitrophenylglutathione ATPase of human erythrocytes. Kinetic constants of the enzyme for the conjugates of glutathione, bile acids and bilirubin are comparable indicating that this ATPase may mediate active transport of all these anionic conjugates in liver.     

Sarcoplasmic reticulum ATPase catalyzes hydrolysis of adenyl-5'-yl imidodiphosphate

Sarcoplasmic reticulum ATPase has been found to cleave the ATP analog adenyl-5'-yl imidodiphosphate in a calcium-dependent reaction. The reaction products were determined by 31P NMR to be inorganic phosphate and adenyl-5'-yl phosphoramidate (AMP-PN). AMP-PNP hydrolysis, like ATP hydrolysis, drives active Ca2+ accumulation by sarcoplasmic reticulum vesicles.     

Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.

To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5'-yl imidodiphosphate and ADP resulted in a significant decrease in the fluorescence intensity of approximately 40 and 30%, respectively. When ATP was added to MIANS-S-1, the fluorescence intensity decreased rapidly by approximately 40%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity increased to approximately 70% of the original value. These results together with model experiments with MIANS-N-acetylcysteine indicate that in the presence of ATP, the MIANS fluorophore attached to SH2 is located in a less hydrophobic environment than is the fluorophore in the absence of ligand and that the hydrolysis of ATP enhances hydrophobicity around the fluorophore. Acrylamide fluorescence quenching studies of MIANS-S-1 confirmed these results, indicating that addition of ATP and ADP to MIANS-S-1 results in an increase in the Stern-Volmer quenching constant of the fluorophore by factors of approximately 3 and 2.5, respectively. The present observations suggest that binding of ATP causes a movement of SH2 toward the protein surface, whereas it goes back into the protein interior after ATP hydrolysis. The results also confirmed previous observations by a chemical cross-linking approach (Hiratsuka, T. (1987) Biochemistry 26, 3168-3173).     

ATP increases the affinity between MutS ATPase domains. Implications for ATP hydrolysis and conformational changes

MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.     

Regulation of the synthesis and hydrolysis of ATP by mitochondrial ATPase. Role of the natural ATPase inhibitor protein

The action of the natural ATPase inhibitor protein of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) on the mechanisms of energy conservation of heart mitochondria has been explored. The synthesis and hydrolysis of ATP and the Pi-ATP exchange reaction were studied in submitochondrial particles that possess the ATPase-inhibitor protein complex in two distinguishable states. In addition to their different rates of hydrolysis, the two states of the complex have been identified from their different accessibility to antibodies directed against the inhibitor protein, and from the different action of antibodies and trypsin on the ATPase activity of the two types of particles studied. The steady state rates of hydrolysis and of the Pi-ATP exchange reaction of the particles are determined by the state in which the ATPase-inhibitor complex exists. Apparently by modifying the rate of one of the steps involved in the catalytic reaction of the ATPase, the inhibitor protein determines the extent to which the enzyme is able to catalyze ATP hydrolysis and the Pi-ATP exchange reaction. This action of the inhibitor protein also reflects the rate at which the particles carry out oxidative phosphorylation.     

Identification of an anion-transport ATPase that catalyzes glutathione conjugate-dependent ATP hydrolysis in canalicular plasma membranes from normal rats and rats with conjugated hyperbilirubinemia (GY mutant).

Rat liver canalicular plasma membranes were found to contain a 37-kDa protein that is immunologically cross-reactive with the dinitrophenyl glutathione-stimulated ATPase previously identified in human tissues. The protein, which was partially purified by affinity chromatography, exhibited ATPase activity dependent on dinitrophenyl glutathione, bilirubin ditaurate, and other dianionic compounds. The localization of this protein in the canalicular membrane and its measured enzymatic activity indicate that it is involved in the transport of glutathione derivatives and other dianionic organic compounds. A rat mutant in which the above transport activities are impaired contained the protein in amounts similar to those in a normal control.     

ATP synthesis at 100 degrees C by an ATPase purified from the hyperthermophilic archaeon Pyrodictium abyssi

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100 degrees C and gains energy by sulfur respiration, with hydrogen as electron donor. From the membranes of this hyperthermophile, an ATPase complex was isolated. The purified enzyme consists of six major polypeptides, the 67, 51, 41, 26 and 22 kDa subunits composing the AF(1) headpiece, and the 7 kDa proteolipid of the AF(0) component. The headpiece of the enzyme restored the formation of ATP during sulfur respiration in membrane vesicles from which it had been removed by low salt treatment. Characteristics of the reconstituted activity suggest that the same enzyme is responsible for ATP formation in untreated membranes. ATP formation was neither sensitive to ionophores and uncouplers, nor to dicyclohexyl carbodiimide, but depended on closed vesicles. Both ATPase activity (up to 2 micromol per min and mg protein) as well as ATP formation (up to 0.4 micromol per min and mg membrane protein) were highest at 100 degrees C. A P/e2 ratio of close to one can be estimated for sulfur respiration with hydrogen. In addition to ATP, autoradiographic detection revealed the formation of high quantities of (33)P(i)-labeled ADP and of another compound not identified so far.     

Studies on ATP hydrolysis in medium for histochemical demonstration of ATPase activity

Summary Nonenzymatic ATP hydrolysis in medium of Wachstein and Meisel for histochemical demonstration of ATPase activity was investigated. In this medium considerable amounts of phosphorus are released without the participation of the enzyme. ATP hydrolysis in Wachstein-Meisel's medium increase with the concentration of Pb⁺⁺ and decrease at its small concentrations. The degree of ATP hydrolysis appeared to increase with increase both temperature and pH. At high concentration of ATP (5.76 mM) the degree of ATP hydrolysis in Wachstein-Meisel's medium is lower than at 1.44 mM ATP. 10.0 mM Ca⁺⁺ or 3.6 mM Fe⁺⁺ speed up ATP hydrolysis after 30- and 60-minute incubation. In the presence of 3.6 mM Co⁺⁺ or 2.6 mM Cu⁺⁺ ATP hydrolysis in Wachstein-Meisel's medium increased throughout the whole period examined. On the contrary, 3.6 mM Fe⁺⁺⁺ decreases ATP hydrolysis in this medium.10.0 mM F^– raises the degree of ATP hydrolysis which is, however, lowered in the presence of 2.5 mM pCMB or 3.6 mM KCN. 2.0 mM cysteine highly inhibits the process of nonenzymatic ATP hydrolysis in Wachstein-Meisel's medium.These data show that the histochemical reaction for ATPase activity in Wachstein-Meisel's medium does not originate exclusively from the hydrolysis of ATP in the presence of Pb⁺⁺, but take rise, above all, as a result of an enzymatic reaction.     

A study of sodium release in the course of ATP hydrolysis by membrane ATPase

Summary With the aid of sodium-sensitive glass electrodes, changes in sodium ion activity were studied in the course of subsequent additions of components required for ATP hydrolysis provided by Na⁺–K⁺-dependent membrane ATPase. Membrane ATPase was obtained from guinea pig kidney cortex. In the presence of ATP, Mg⁺⁺ and Na⁺ in media, the addition of K⁺ caused an increase in Na⁺ activity. The omission of ATP or its substitution by ADP as well as the addition of Ca⁺⁺ to the media eliminated the above-mentioned increase of Na⁺ activity. Quabain did not affect Na⁺ release caused by the addition of K⁺, although it significantly inhibited ATPase activity of the preparation. The data obtained were considered to be a direct indication of ion exchange during the course of membrane ATPase reaction. This ion-exchange stage of the reaction is not inhibited by ouabain. The ratio of sodium ions released per one inorganic phosphate formed in the course of the reaction was found to be much higher than that established for transporting membranes of intact cells. A possible cause of this difference is discussed.     

Structural dynamics in F1ATPase during the first reaction cycle of ATP hydrolysis

The velocity of ATP hydrolysis, catalyzed by purified F1ATPase from Micrococcus luteus, was decelerated on decreasing the temperature. At 13 degrees C one reaction cycle is completed after 20 s. Hydrolysis was triggered upon rapid mixing of the enzyme with ATP. During the first reaction cycle, succeeding structural alterations of the F1ATPase were traced by time resolved X-ray scattering. The scattering spectra obtained from consecutive intervals of 1 s, revealed the F1ATPase to pass a conformational state exhibiting an expanded (6%) molecular shape. The expanded state was observed between 45% and 65% of the time required to complete the reaction cycle. This points out a conformational pulsation during ATP hydrolysis.     

The human escort protein Hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

The 44P subunit of the T4 DNA polymerase accessory protein complex catalyzes ATP hydrolysis

The genes encoding all three T4 DNA polymerase accessory proteins have been cloned into overexpression plasmids. Induction of cells harboring these plasmids results in the synthesis of each accessory protein at levels that approach 10% of the total cellular protein. The solubility of the accessory proteins after induction at 42 degrees C ranges from about 60% to greater than 95%. A plasmid that allows overexpression of the 44P/62P complex has been manipulated further to overexpress selectively the 44P subunit without 62P, permitting us to assess how each subunit contributes to the properties of the 44P/62P complex. A comparison of 44P and 44P/62P by conventional hydrodynamic techniques shows that 44P forms a subcomplex nearly as large as the 44P/62P complex. In addition, 44P catalyzes DNA-dependent ATP hydrolysis with a specific activity similar to that of the 44P/62P ATPase. However, unlike the 44P/62P complex, the ATPase activity of 44P alone is only slightly stimulated by 45P. This suggests that one role of the 62P subunit is to facilitate a productive interaction of 44P and 45P.     

Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation

Membrane-associated ATPase constitutes an essential element common to all secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild-type XpsE. Yet the mutant XpsE(R286A) is non-functional in protein secretion via T2SS. Detailed analyses indicated that the mutant XpsE(R286A) lost the ability co-ordinating the N- and C-domain of XpsE. Without significantly influencing XpsE binding affinity with ATP or its oligomerization, R286A mutation however, caused XpsE lose the ability to associate with the cytoplasmic membrane via XpsL(N). As a consequence, ATP hydrolysis by XpsE was uncoupled from protein secretion. Because R286 is highly conserved among members of the secretion NTPase superfamily, we speculate that its equivalent in other homologues may also play a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion system.     

Active transport of ATP and presence of a vanadate-sensitive membrane-bound ATPase in Mycobacterium leprae.

It is not known how Mycobacterium leprae obtains energy for survival and growth in the host tissues; the organism does not grow in vitro. In the studies reported here, M. leprae incorporated labelled ATP, which was blocked by cyanide, unlabelled ATP or ADP, but not by adenosine or Pi. It seems that the organism takes up unhydrolysed ATP by an active transport process. The bacterium contained a membrane-bound, vanadate-sensitive E1 E2-ATPase (which creates a transmembrane potential driving transport of solutes into cells). The enzyme was not inhibited by N-ethylmaleimide, suggesting that it is not an F0F1-ATPase which catalyses ATP synthesis. Apparently, M. leprae derives energy-rich compounds from the host cell.     

Legionella micdadei phosphatase catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate in human neutrophils

The legionellae are facultative intracellular bacterial pathogens which multiply in host phagocytes. Legionella micdadei cells contain an acid phosphatase (ACP2) which blocks superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe (fMLP) [A. K. Saha, et al. (1985) Arch. Biochem. Biophys. 243, 150-160]. In the present study, we have purified the Legionella phosphatase to homogeneity as indicated by the finding of a single 68,000-Da band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We explored the possibility that ACP2 acts by interfering with polyphosphoinositide hydrolysis and the production of the intracellular second messengers, inositol trisphosphate (IP3) and diacylglycerol, following neutrophil stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) was hydrolyzed rapidly by ACP2 in vitro. The rate of hydrolysis of PIP2 was higher at pH 7.0 (Km 2.0 microM; 4 X 10(3) units/mg protein; 1 unit equals 1 nmol of Pi released/h) than at lower pH. IP3 was also a good substrate for ACP2 in vitro. When human neutrophil phosphoinositides were prelabeled with 32Pi, subsequent incubation with ACP2 resulted in an 85% loss of the labeled PIP2 over 2 h. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by ACP2-treated cells was reduced by 44%. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. These results indicate that the Legionella phosphatase may compromise the neutrophils' microbicidal response to the organism by hydrolyzing PIP2, the progenitor of IP3 and diacylglycerol, and by hydrolyzing IP3 itself.