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)

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could be unambiguously assigned to the switch II helix (residues 475-507) whose N-terminus lies 14.2 A from Cys 540 and would include the ligand-responsive cleavage site. This motif is thought to be crucial for the transmission of sub-nanometer structural changes at the ATPase site to both the actin site and the lever arm domain during energy transduction. Our study illustrates this novel, actin site-specific chemical proteolysis of S-1 as a direct probe of the switch II helix conformational transitions in solution most likely associated with the skeletal cross-bridge cycle.
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PMID:Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site. 1050 94

FtsH, a membrane-bound and ATP-dependent protease of Escherichia coli, is involved in degradation of some of uncomplexed integral membrane proteins and short-lived cytoplasmic proteins. It is composed of an N-terminal membrane-spanning region and a following large cytoplasmic domain that contains ATPase and protease active sites. In the present study, it was found that FtsH undergoes C-terminal processing in vivo. The processing was blocked by loss of function mutations of FtsH. Purified FtsH-His(6)-Myc, a C-terminally tagged derivative of FtsH, was self-processed in vitro. This in vitro processing was observed only in the presence of ATP and not in the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP). Moreover, such processing did not occur in the case of the ATPase motif mutant protein. These results indicated that this processing is a self-catalyzed reaction that needs ATP hydrolysis. Mutations in the hflKC genes that encode a possible modulator of FtsH, and the growth phase of the cells as well, affected the processing. Complementation experiments with genetically constructed variants suggested that both the processed and the unprocessed forms of FtsH are functional. The cleavage was found to occur between Met-640 and Ser-641, removing a heptapeptide from the C-terminus of FtsH. Systematic mutational analyses of Met-640 and Ser-641 revealed preferences for positively charged and hydrophobic amino acid residues at these positions for processing. This cleavage specificity may be shared by the self-cleavage and the substrate-cleavage reactions of this protease.
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PMID:Self-processing of FtsH and its implication for the cleavage specificity of this protease. 1051 25

Both the secAcsR11 and DeltasecG::kan mutations cause cold-sensitive growth, although the growth defect due to the latter mutation occurs in a strain-specific manner. Overexpression of pgsA encoding phosphatidylglycerophosphate synthase suppresses the growth defects of the two mutants. We investigated the mechanism underlying the pgsA-dependent suppression of the two mutations using purified mutant SecA and inverted membrane vesicles (IMVs) prepared from pgsA-overexpressing cells. The acidic phospholipid content increased by about 10% upon pgsA overexpression. This increase resulted in the stimulation of proOmpA translocation only when mutant SecA or SecG-depleted IMVs were used. The translocation-coupled ATPase activity of SecA was significantly defective with the mutant SecA or SecG-depleted IMVs, but it recovered to a near normal level when the acidic phospholipid level was increased. The stimulation of ATPase activity was observed only at low temperature. The steady-state level of membrane-inserted SecA was low with the mutant SecA or SecG-depleted IMVs, and it decreased further upon the increase in the acidic phospholipid content. However, the level of SecA insertion markedly increased upon the inhibition of SecA deinsertion by the addition of beta,gamma-imido adenosine 5'-triphosphate (AMP-PNP), especially with IMVs containing increased levels of acidic phospholipids. These results indicate that the increase in the level of acidic phospholipids stimulates the SecA cycle in the two mutants by facilitating both the insertion and deinsertion of SecA.
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PMID:Increases in acidic phospholipid contents specifically restore protein translocation in a cold-sensitive secA or secG null mutant. 1052

In the crystal structure of the bovine heart mitochondrial F(1)-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded beta subunits, one with MgAMP-PNP bound to the catalytic site (beta(T)) and the other with MgADP bound (beta(D)) have closed conformations. The empty beta subunit (beta(E)) has an open conformation. In beta(T) and beta(D), the distance between the carboxylate of beta-Asp(315) and the guanidinium of beta-Arg(337) is 3.0-4.0 A. These side chains are at least 10 A apart in beta(E). The alpha(3)(betaD311C/R333C)(3)gamma subcomplex of TF(1) with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl(2) with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[(14)C]acetamide after prolonged treatment with CuCl(2) in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a beta subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F(1) are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.
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PMID:Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously. 1053 37

The isolated H(+)-ATPase from Escherichia coli (EF(0)F(1)) was investigated by electron microscopy of samples of negatively stained monodisperse molecules, followed by single-particle image processing. The resulting three-dimensional maps showed that the F(1)-part is connected by a prominent stalk to a more peripheral part of F(0). The F(1)-part showed stain-accessible cavities inside. In three-dimensional maps from selected particles, a second stalk could be detected which was thinner than the main stalk and is thought to correspond to the stator.Three-dimensional maps of the enzyme in the absence and in the presence of the substrate analogue adenyl-beta, gamma-imidodiphosphate (AMP-PNP) were calculated. Upon binding of AMP-PNP the three-dimensional maps showed no significant changes in the F(0)-part of EF(0)F(1), whereas a major conformational change in the F(1)-part was observed. (1) The diameter of the F(1)-part decreased upon binding of AMP-PNP mainly in the upper half of F(1). (2) Enzyme particles prepared in the presence of AMP-PNP had a pointed cap at the top of the F(1)-part which was missing in its absence. (3) The stain-accessible cavity inside the F(1)-part altered its pattern significantly.
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PMID:Direct visualisation of conformational changes in EF(0)F(1) by electron microscopy. 1066

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.
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PMID:Structural changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding studied by fourier transform infrared spectroscopy. 1069 37

In Scrobicularia plana testis, a nuclear acid phosphatase (ACPase) activity was detected in mid and late spermatids with the improved Gomori-chloride procedure. Lead deposits were first observed in mid spermatids at focal points over condensed chromatin strands, increasing in density as chromatin further condensated. In late spermiogenesis, lead deposits became concentrated between chromatin aggregates, and after total DNA compaction were transfered to the nuclear periphery and then shed into the cytoplasm. The specificity of the nuclear ACPase was tested against different pH values (3.9, 7.2, 7.8, 9.0), substrates (TPP, IDP, TMP, p-NCS, ATP, GTP, AMP, ADP, AMP-PNP) and inhibitors (NaF, levamisole, Zn, vanadate, theophylline). To further specify the nature of this nuclear ACPase, other enzymes were comparatively studied at their optimal pH values and at pH 5.0: nucleoside-diphosphatase, thiamin-pyrophosphatase, inorganic trimetaphosphatase, lysosomal arylsulfatases A and B, ATPase, GTPase, 5'-nucleotidase, adenylate kinase, and adenylate cyclase. Several other controls were introduced to exclude artefactual deposits induced by lead ions and tissue molecules. The results showed that the enzyme has an optimal pH at 5.0, a high specific affinity for beta-GP, and is inhibited by NaF, which suggests that it behaves as a type B-ACPase, and all controls demonstrated the specificity of the enzymic activity. Because lead deposits were specifically and temporally associated with spermatid chromatin condensation, when DNA and RNA synthesis, histones, phosphoproteins and RNA molecules strongly decrease, it is possible to suggest that the nuclear ACPase could be associated with DNA processing during chromatin compaction or involved in the hydrolysis of 2' and 3' nucleotides resulting from nuclear RNase action during RNA degradation.
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PMID:Chromatin condensation during Scrobicularia plana spermiogenesis: a controlled and comparative enzymatic ultracytochemical study. 1079 22

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.
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PMID:The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains. 1094 21

Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.
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PMID:Identification and characterization of a novel microtubule-based motor associated with membranous organelles in tobacco pollen tubes. 1100 43

In the presence of ascorbate/H(2)O(2), ATP-Fe(2+) or AMP-PNP-Fe(2+) complexes act as affinity cleavage reagents, mediating selective cleavage of the alpha subunit of Na,K-ATPase at high affinity ATP-Mg(2+) sites. The cleavages reveal contact points of Fe(2+) or Mg(2+) ions. In E(1) and E(1)Na conformations, two major cleavages are detected within the conserved (708)TGDGVNDSPALKK sequence (at V712 and nearby), and one (E(1)Na) or two (E(1)) minor cleavages near V440. In media containing sodium and ATP, Fe(2+) substitutes for Mg(2+) in activating phosphorylation and ATP hydrolysis. In the E(1)P conformation, cleavages are the same as in E(1). Fe(2+) is not bound tightly. By contrast, in the E(2)P conformation, the pattern is different. A major cleavage occurs near the conserved sequence (212)TGES, whereas those in TGDGVNDSPALKK are less prominent. Fe(2+) is bound very tightly. On E(2)P hydrolysis, the Fe(2+) dissociates. The results are consistent with E(1)<-->E(2) conformation-dependent movements of cytoplasmic domains and sites for P(i) and Mg(2+) ions, inferred from previous Fe-cleavage experiments. Furthermore, these concepts fit well with the crystal structure of Ca-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature (London) 405, 647-655]. Altered ligation of Mg(2+) ions in E(2)P may be crucial in facilitating nucleophilic attack of water on the OP bond. Mg(2+) ions may play a similar role in all P-type pumps. As affinity cleavage reagents, ATP-Fe(2+) or other nucleotide-Fe(2+) complexes could be widely used to investigate nucleotide binding proteins.
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PMID:The complex ATP-Fe(2+) serves as a specific affinity cleavage reagent in ATP-Mg(2+) sites of Na,K-ATPase: altered ligation of Fe(2+) (Mg(2+)) ions accompanies the E(1)-->E(2) conformational change. 1103 1


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