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 processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.
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PMID:Alternating site mechanism of the kinesin ATPase. 945 68

The knowledge about the structure and function of the protein families responsible for cGMP synthesis and metabolic conversion has grown vastly the last years, whereas little is known about proteins that account for the cellular export of cGMP. In the present study, we have employed a model with inside-out vesicles prepared from human erythrocytes to characterize modulation and regulation of cellular cGMP extrusion. The active transport was saturable (Km of 2.4 +/- 0.2 microM, mean +/- SEM, n = 3) and coupled to ATP hydrolysis since no accumulation was detected in the presence of ATP-gamma-S and AMP-PNP. The observation that 100 microM of cAMP caused a minimal inhibition (14.4 +/- 0.3%) of active cGMP transport showed that the extrusion system for cGMP was not shared with cAMP, but a competitive interaction occurred for the ATP-independent association to the inside out vesicles. In contrast, the lowest, but physiological relevant cAMP concentrations (0.1-5 microM) stimulated the active cGMP transport with 30-35%, an observation that suggests cAMP as an allosteric regulator of the cGMP transporter. Several well-known modulators of other energy-requiring membrane transport systems caused a competitive and concentration-dependent inhibition, including verapamil (Ki = 13.0 +/- 2.4 microM), forskolin (Ki = 13.5 +/- 1.4 microM) and probenecid (Ki = 27.0 +/- 1.3 microM). Progesterone, which was the most potent inhibitor (Ki = 2.2 +/- 0.3 microM), interacted with the active cGMP transport in a noncompetitive manner. The highest concentration (100 microM) of IBMX and theophylline reduced the active cGMP uptake with 29.5 +/- 1.9% and 21.6 +/- 2.1%, respectively. None of these substances interfered with the association of cGMP to the vesicles in absence of ATP. The present results show that human erythrocytes possess a cell membrane cGMP transporter which is coupled to an ATPase. Its activity is regulated by cAMP in an apparent allosteric manner and inhibited by substances previously known to interact with other membrane transport systems.
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PMID:Cyclic AMP stimulates the cyclic GMP egression pump in human erythrocytes: effects of probenecid, verapamil, progesterone, theophylline, IBMX, forskolin, and cyclic AMP on cyclic GMP uptake and association to inside-out vesicles. 945 9

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolising cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Structure of the two enzyme forms and the effects of beta, gamma-imidoadenosine 5'-triphosphate (AMP-PNP) and of diethylstilbestrol (DES) thereon were analysed by FT-IR spectroscopy. IR spectra revealed the presence of two populations of alpha-helices with different exposure to the solvent in both the A-ATPase and B-ATPase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two alpha-helix populations. Thermal denaturation experiments suggested a more stable structure in the B-form than in the A-form. AMP-PNP stabilised the A-ATPase structure while DES destabilised both enzyme forms. IR spectra showed that 60% of the amide hydrogens were exchanged for deuterium in both forms at 20 degrees C. The remaining 40% were exchanged at higher temperatures. The maximum amount of H/D exchange was observed at 50-55 degrees C for both enzyme forms, while in the presence of DES it was observed at lower temperatures. The data do not contradict the possibility that the activation of H(+)-ATPase is due to the C-terminus of the enzyme dissociating from the ATP-binding site which is covered by it in the less active form.
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PMID:Structure of yeast plasma membrane H(+)-ATPase: comparison of activated and basal-level enzyme forms. 952 79

Cell membrane fluctuations (CMF) of human erythrocytes, measured by point dark field microscopy, were shown to depend, to a large extent, on intracellular MgATP (Levin, S.V., and R. Korenstein. 1991. Biophys. J. 60:733-737). The present study extends that investigation and associates CMF with F-actin's ATPase activity. MgATP was found to reconstitute CMF in red blood cell (RBC) ghosts and RBC skeletons to their levels in intact RBCs, with an apparent Kd of 0.29 mM. However, neither non-hydrolyzable ATP analogues (AMP-PNP, ATPgammaS) nor hydrolyzable ones (ITP, GTP), were able to elevate CMF levels. The inhibition of ATPase activity associated with the RBC's skeleton, carried out either by the omission of the MgATP substrate or by the use of several inhibitors (vanadate, phalloidin, and DNase I), resulted in a strong decrease of CMF. We suggest that the actin's ATPase, located at the pointed end of the short actin filament, is responsible for the MgATP stimulation of CMF in RBCs.
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PMID:Mechanical fluctuations of the membrane-skeleton are dependent on F-actin ATPase in human erythrocytes. 964 48

The plasma membrane H+-ATPase of yeast assumes distinct conformational states during its catalytic cycle. To better understand structural changes in the LOOP1 domain, a catalytically important cytoplasmic loop segment linking transmembrane segments 2 and 3, surface epitopes were examined at different stages of catalysis. A polyclonal rabbit antibody was prepared to a fusion protein consisting of LOOP1 and the maltose binding protein. This antibody was affinity-purified to produce a LOOP1-specific fraction that could be used in competition enzyme-linked immunosorbent assays to assess surface exposure of the LOOP1 epitopes. It was found that in an E1 conformation stabilized with either adenosine 5'-(beta,gamma -imino)triphosphate (AMP-PNP) or ADP, less than 10% of the LOOP1 epitopes were accessible on native enzyme. However, when the enzyme was stabilized in an E2-state with ATP plus vanadate, approximately 40% of the surface epitopes on LOOP1 became accessible to antibody. The remaining 60% of the LOOP1 epitopes were fully occluded in the native enzyme and never showed surface exposure. Enzyme-linked immunosorbent assays utilizing fusion proteins consisting of LOOP1 subdomains demonstrated that all of the available epitopes were contained in the beta-strand region (Glu-195-- Val-267) of LOOP1. The epitopes that were differentially exposed during catalysis were included in regions upstream and downstream of the highly conserved TGES sequence. Our results suggest that during catalysis either the beta-strand region of LOOP1 or an interacting domain undergoes substantial structural rearrangement that facilitates epitope exposure.
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PMID:Differential exposure of surface epitopes in the beta-strand region of LOOP1 of the yeast H+-ATPase during catalysis. 966 Jul 92

To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta, gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.
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PMID:Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP. 975 27

The shape and subunit arrangement of the Escherichia coli F1 ATPase (ECF1 ATPase) was investigated by synchrotron radiation x-ray solution scattering. The radius of gyration and the maximum dimension of the enzyme complex are 4.61 +/- 0.03 nm and 15.5 +/- 0.05 nm, respectively. The shape of the complex was determined ab initio from the scattering data at a resolution of 3 nm, which allowed unequivocal identification of the volume occupied by the alpha3beta3 subassembly and further positioning of the atomic models of the smaller subunits. The delta subunit was positioned near the bottom of the alpha3beta3 hexamer in a location consistent with a beta-delta disulfide formation in the mutant ECF1 ATPase, betaY331W:betaY381C:epsilonS108C, when MgADP is bound to the enzyme. The position and orientation of the epsilon subunit were found by interactively fitting the solution scattering data to maintain connection of the two-helix hairpin with the alpha3beta3 complex and binding of the beta-sandwich domain to the gamma subunit. Nucleotide-dependent changes of the delta subunit were investigated by stopped-flow fluorescence technique at 12 degrees C using N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) as a label. Fluorescence quenching monitored after addition of MgATP was rapid [k = 6.6 s-1] and then remained constant. Binding of MgADP and the noncleavable nucleotide analog AMP . PNP caused an initial fluorescent quenching followed by a slower decay back to the original level. This suggests that the delta subunit undergoes conformational changes and/or rearrangements in the ECF1 ATPase during ATP hydrolysis.
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PMID:A model of the quaternary structure of the Escherichia coli F1 ATPase from X-ray solution scattering and evidence for structural changes in the delta subunit during ATP hydrolysis. 978 16

Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.
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PMID:Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains. 985 72

The conformational properties of the molecular chaperone GroEL in the presence of ATP, its non-hydrolyzable analog 5'-adenylimidodiphosphate (AMP-PNP), and ADP have been analyzed by differential scanning calorimetry (DSC), Fourier-transform infra-red (FT-IR) and fluorescence spectroscopy. Nucleotide binding to one ring promotes a decrease in the Tm value of the GroEL thermal transition that is reversed when both rings are filled with nucleotide, indicating that the sequential occupation of the two protein rings by these nucleotides has different effects on the GroEL thermal denaturation process. In addition, ATP induces a conformational change in GroEL characterized by (a) the appearance of a reversible low temperature endotherm in the DSC profiles of the protein, and (b) an enhanced binding of the hydrophobic probe 8-anilino-naphthalene-1-sulfonate (ANS), which strictly depends on ATP hydrolysis. The similar sensitivity to K+ of the temperature range where activation of the GroEL ATPase activity, the low temperature endotherm, and the increase of the ANS fluorescence are abserved strongly indicates the existence of a conformational state of GroEL during ATP hydrolysis, different from that generated on ADP or AMP-PNP binding. To achieve this intermediate conformation, GroEL mainly modifies its tertiary and quaternary structures, leading to an increased exposure of hydrophobic surfaces, with minor rearrangements of its secondary structure.
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PMID:ATP hydrolysis induces an intermediate conformational state in GroEL. 991 13

N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd motor-stalk protein MC1 (modified claret 1) was treated with varying concentrations of NEM (0-10 mM) and cosedimentation and ATPase assays were used to assess the effects of modification on MC1 interactions with MTs. In the cosedimentation assay, treatment with </=0.1 mM NEM enhanced MC1 binding to MTs in the presence of MgATP but had no effect on MC1 binding to MTs in the presence of MgAMP-PNP. In comparison, treatment with >/=0.5 mM NEM induced aggregation of MC1 and resulted in sedimentation of the motor in the absence of MTs. NEM modification had no effect on the basal ATPase rate but produced a decrease in the MT-stimulated ATPase rate. Labeling of MC1 with [3H]NEM indicated that enhanced MT binding was associated with an average labeling of 1 Cys residue per MC1 polypeptide, while aggregation was associated with an average labeling of 2 Cys residues per MC1 polypeptide. Protein digestion, structural analysis, and mass spectrometry indicate that modification of Cys313 or Cys324 in the stalk domain is correlated with enhanced binding of MC1 to MTs. These results suggest that NEM enhances Ncd binding to MTs by disruption of neck and/or stalk function and demonstrate the importance of this region in motor function.
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PMID:N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain. 1045 70


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