Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chaperonin proteins, GroEL14 and GroES7, inhibit protein aggregation and assist in protein folding in a potassium/ATP-dependent manner. In vitro, assays for chaperonin activity typically involve adding a denatured substrate protein to the chaperonins and measuring the appearance of correctly folded substrate protein. The influence of denaturant is generally ignored. Low concentrations of guanidinium chloride (< 100 mM) had a profound effect on the activity/structure of the chaperonins. Guanidinium decreased the ATPase activity of GroEL and attenuated the inhibition of GroEL ATP hydrolysis by GroES. The stable, asymmetric chaperonin complex which forms in the presence of GroES and ADP (GroES7.ADP7.GroEL7-GroEL7) rapidly dissociated upon addition of 80 mM guandinium chloride. Dissociation was enhanced at high ionic strength, but rapid dissociation was guanidinium-specific. Accelerated release of the GroES from the complex was also demonstrated. Unfolded proteins alone had no effect on complex stability. Residual guanidinium depressed the rate of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase (Rubisco) folding; an increased aggregation rate also decreased the yield of folded Rubisco. Chaperonin-assisted folding is therefore best studied using proteins denatured by means other than guanidinium chloride.
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PMID:Stability of the asymmetric Escherichia coli chaperonin complex. Guanidine chloride causes rapid dissociation. 789 Jun 52

Previous work on the role of occluded Rb+ (a K+ substitute) in the reaction cycle of (Na+ + K+)-ATPase has focused on the kinetics of the dissociation of the enzyme-Rb+ complex at 20-24 degrees C. Doing experiments at 4 degrees C, we have made the following observations on the equilibrium binding levels and the kinetics of binding and release of Rb+. 1) The plot of bound Rb+ as a function of [Rb+] showed occupancy of high affinity sites, followed by binding to sites of lower affinity. The estimated number of Rb+ sites/active site was two to three, but a higher number was not ruled out. Release of bound Rb+ was slow and not monoexponential, the major portion being in a pool with a half-life of 4-5 h. Dissociation curves were identical at different levels of site occupancy. Rb+ binding also had fast and slow phases, requiring about 24 h to reach steady state at vastly different [Rb+]. These data suggest that (a) Rb+ occlusion sites are confined within the protein matrix and connected to the medium by narrow access channels that are heterogeneous in size, and (b) channel heterogeneity is distinct from differences in occlusion site affinities. 2) ATP, at a low affinity allosteric site, had no significant effect on the maximal level of bound Rb+ at any [Rb+], but it accelerated both the fast and the slow phases of Rb+ binding and release, and it increased the ratio of fast to slow phases. Evidently, ATP activates the channels (lowers the energy barrier for access) without altering binding site affinities. 3) Na+ was a competitive inhibitor of Rb+ at the occluded sites, but it also acted at an allosteric site to activate the access channels. Rb+ and K+ also had allosteric effects: although they did not affect the access channels directly, they blocked the allosteric effect of Na+. 4) Ouabain was an access channel inhibitor. It reduced the rates of binding and release of Rb+, blocked channel activation by ATP and Na+, but seemed to have no effect on the events at the occluded sites. The existence of heterogeneous access channels to the ion transport sites and the demonstration of channel regulation by the physiological ligands of the enzyme suggest the necessity of the inclusion of such allosteric mechanisms in the reaction cycle of (Na+ + K+)-ATPase.
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PMID:Allosteric regulation of the access channels to the Rb+ occlusion sites of (Na+ + K+)-ATPase. 838 24

The RuvAB, RuvC and RecG proteins of Escherichia coli process intermediates in recombination and DNA repair into mature products. RuvAB and RecG catalyse branch migration of Holliday junctions, while RuvC resolves these structures by nuclease cleavage around the point of strand exchange. The overlap between RuvAB and RecG was investigated using synthetic X- and Y-junctions. RuvAB is a complex of RuvA and RuvB, with RuvA providing the DNA binding subunit and RuvB the ATPase activity that drives branch migration. Both RuvA and RecG form defined complexes with each of the junctions. The gel mobilities of these complexes suggests that the X-junction attracts two tetramers of RuvA, but mainly monomers of RecG. Dissociation of the junction in the presence of ATP requires high levels of RuvAB. RecG is shown to have a much higher specific activity to the extent that very little of this protein would be required to match RuvAB in vivo. Both proteins also dissociate a Y-junction, which is consistent with helicase activity. However, RecG shows no ability to unwind more conventional substrates and the suggestion is made that its helicase activity is directed towards specific DNA structures such as junctions.
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PMID:Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli. 838 95

Incubation of the purified F1F0-ATPase of Propionigenium modestum with dicyclohexylcarbodiimide (DCCD) led to inactivation of the enzyme in a strongly pH-dependent manner. Rapid inactivation occurred at pH 5-7, while the increase of the pH from 7 to 9 resulted in a continuous reduction of the inactivation rate. In the presence of Na+ ions, the ATPase was specifically protected from inactivation by DCCD. The protective effect of Na+ was most pronounced at pH 9.0 and less significant at pH 7.0. In addition to Na+, Li+ also protected the ATPase from inactivation by DCCD, but approximately 10 times higher concentrations were required for the same effect. Similarly, the Na+ concentration causing half-maximal stimulation of ATPase activity was about 10 times below the Li+ concentration required for the same activation. It is concluded from these results that a binding site is present for Na+ or Li+ on the enzyme with an about 10 times lower affinity for the latter alkali ion, which when occupied stimulates ATPase activity and protects it from inactivation by DCCD. Inactivation of ATPase activity by DCCD correlated well with a specific labeling of subunit c of the enzyme in the presence of the [14C]DCCD derivative. Like ATPase inactivation, the labeling was promoted by more acidic pH values and inhibited by Na+ ions. We suggest from these data that the DCCD-reactive amino acid residue of subunit c (most likely Glu-65) must be protonated for the reaction with the carbodiimide and provides the Na(+)-binding site in its deprotonated state. Dissociation of the carboxylic acid (at high pH) and binding of Na+ ions to the carboxylate thus abolish the reactivity toward DCCD.
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PMID:Specific protection by Na+ or Li+ of the F1F0-ATPase of Propionigenium modestum from the reaction with dicyclohexylcarbodiimide. 839 53

The role of prolonged electrical stimulation on sarcoplasmic reticulum (SR) Ca2+ sequestration measured in vitro and muscle energy status in fast white and red skeletal muscle was investigated. Fatigue was induced by 90 min intermittent 10-Hz stimulation of rat gastrocnemius muscle, which led to reductions (p < 0.05) in ATP, creatine phosphate, and glycogen of 16, 55, and 49%, respectively, compared with non-stimulated muscle. Stimulation also resulted in increases (p < 0.05) in muscle lactate, creatine, Pi, total ADP, total AMP, IMP, and inosine. Calculated free ADP (ADPf) and free AMP (AMPf) were elevated 3- and 15-fold, respectively. No differences were found in the metabolic response between tissues obtained from the white (WG) and red (RG) regions of the gastrocnemius. No significant reductions is SR Ca2+ ATPase activity were observed in homogenate (HOM) or a crude SR fraction (CM) from WG or RG muscle following exercise. Maximum Ca2+ uptake in HOM and CM preparations was similar in control (C) and stimulated (St) muscles. However, Ca2+ uptake at 400 nM free Ca2+ was significantly reduced in CM from RG (0.108 +/- 0.04 to 0.076 +/- 0.02 mumol.mg-1 protein.min-1 in RG - C and RG - St, respectively). Collectively, these data suggest that reductions in muscle energy status are dissociated from changes in SR Ca2+ ATPase activity in vitro but are related to Ca2+ uptake at physiological free [Ca2+ bd in fractionated SR from highly oxidative muscle. Dissociation of SR Ca2+ ATPase activity from Ca2+ uptake may reflect differences in the mechanisms evaluated by these techniques.
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PMID:Effects of prolonged low frequency stimulation on skeletal muscle sarcoplasmic reticulum. 856 84

At least part of the gamma subunit of the catalytic portion of the chloroplast ATP synthase (CF1) is present in the middle of the alpha3beta3 heterohexamer. Interactions of the alpha/beta subunits with the gamma subunit stabilize the hexameric structure. Surprisingly, neither reduction of the gamma disulfide nor selective proteolysis of alpha, beta and gamma affects the thermal stability of EDTA-treated CF1 preparations, as determined by differential scanning calorimetry. Dissociation of the enzyme in the cold may be monitored by loss of the ATPase activity of CF1 subunit depleted of its epsilon subunit [CF1(-epsilon)]. The rate of cold inactivation of ATPase activity of reduced and alkylated CF1(-epsilon) treated with trypsin in solution was much faster than that CF1(-epsilon)(8.1 versus 38.7 min, respectively, for 50% loss of activity). The increased cold liability of the trypsin-treated enzyme was not a consequence of the cleavage of the gamma. CF1 incubated with trypsin under conditions in which gamma is not cleaved was as cold labile as CF1 with cleaved gamma. Instead, loss of the delta subunit and a few residues from the C-terminal of the beta subunits were responsible for the increased cold liability of the enzyme.
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PMID:Structural stability of chloroplast coupling factor 1 determined by differential scanning calorimetry and cold inactivation. 866 76

P-Glycoprotein is a member of the ABC superfamily of membrane transporters, and functions as an ATP-driven active efflux pump for natural products and chemotherapeutic drugs. Overexpression of P-glycoprotein is a major cause of multidrug resistance in human cancers. Sulfhydryl modification agents are known to inactivate both P-glycoprotein ATPase activity and transport function. In the present study, P-glycoprotein purified from CHRB30 cells was covalently labeled at two conserved Cys residues, one within each of the nucleotide binding domains, using 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). MIANS modification inactivated P-glycoprotein ATPase function, in a concentration-dependent fashion. Increasing concentrations of ATP blocked MIANS labeling with an IC50 of 0.37 mM (similar to the KM for ATP hydrolysis), which suggests that the label is located close to the site of ATP binding within the nucleotide binding domain. A blue shift in the fluorescence spectrum of MIANS bound to P-glycoprotein indicated that the labeled Cys residues are situated in a nonpolar environment. MIANS-labeled P-glycoprotein was still able to bind ATP, as demonstrated by quenching of the fluorescence, with a Kd of 0.46 mM. Addition of a variety of drugs and chemosensitizers to MIANS-labeled P-glycoprotein led to substantial quenching of the probe fluorescence within the nucleotide binding domains. Dissociation constants for drug binding measured by fluorescence quenching were in the range of 0.77 microM for vinblastine to 158 microM for colchicine. Quenching by ATP and drugs was independent and additive, suggesting that each produces a defined change in the protein. The rate of MIANS labeling of Pgp was reduced in the presence of drugs and chemosensitizers, implying that a long-range conformational change arises from drug binding which alters the accessibility of the nucleotide binding domains to MIANS. These results suggest that there is conformational communication between the drug binding site(s) of P-glycoprotein and the ATPase catalytic sites within the nucleotide binding domains.
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PMID:Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains. 879 69

The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.
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PMID:Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump. 891 88

Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in carbamoyl phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze carbamoyl phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of carbamoyl phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.
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PMID:Pressure-induced dissociation of carbamoyl-phosphate synthetase domains. The catalytically active form is dimeric. 960 18

The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore. Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.
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PMID:Reversible association between the V1 and V0 domains of yeast vacuolar H+-ATPase is an unconventional glucose-induced effect. 981 93


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