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)

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.
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PMID:Purification and characterization of a DNA helicase from pea chloroplast that translocates in the 3'-to-5' direction. 866 52

ATP-dependent RNA helicases from the DEAD box family of proteins are involved in a number of RNA processing and utilization events. An3 protein from Xenopus laevis is an RNA helicase of the DEAD box family of proteins. An3 is synthesized by a mRNA that is localized to one end of Xenopus laevis oocytes. An3 protein is found in the nucleus of ooctes, and more specifically, during the middle stages of oocyte development, with extra nucleoli that contain amplified copies of rRNA genes in the nucleolus. By expressing glutathione-S-transferase:An3 fusion proteins in E. coli, sufficient amounts of An3 protein were isolated to examine its enzymatic activities. ATPase activity, NTP substrate range and RNA helicase activity were tested. An3 protein ATPase activity was evident but not stimulated by any of a variety of RNA tested. An3 protein was able to resolve the duplex formed by an in vitro substrate, in the presence of ATP or dATP. An3 required both 3' and 5' single-stranded regions of RNA flanking the RNA duplex it resolves.
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PMID:An3 protein encoded by a localized maternal mRNA in Xenopus laevis is an ATPase with substrate-specific RNA helicase activity. 904 87

The expression of genes transcribed by the RNA polymerase with the alternative sigma factor sigma 54 (E sigma 54) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter. These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis. The best characterized proteins of this family of regulators are NtrC and NifA, which activate genes required for ammonia assimilation and nitrogen fixation, respectively. In a recent IRBM course (@ontiers of protein structure prediction," IRBM, Pomezia, Italy, 1995; see web site http://www.mrc-cpe.cam.uk/irbm-course95/), one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold. This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology. In this work, we present new data that support the previous conclusion. The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alpha/beta topology. The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence. Finally, and most importantly, three of five reported residue alterations that impair the Central domain. ATPase activity of the E sigma 54 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotide-binding in the mononucleotide-binding proteins. Furthermore, the known residue substitution that alter the function of the E sigma 54 activators, leaving intact the Central domain ATPase activity, are mapped on region proposed to play an equivalent role as the effector region of the GTPase superfamily.
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PMID:A proposed architecture for the central domain of the bacterial enhancer-binding proteins based on secondary structure prediction and fold recognition. 907 Apr 37

Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.
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PMID:Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein. 956 9

The mechanical behavior of skinned rabbit psoas muscle fiber contractions and in vitro motility of F-actin (Vf) have been examined using ATP, CTP, UTP, or their 2-deoxy forms (collectively designated as nucleotide triphosphates or NTPs) as contractile substrates. Measurements of actin-activated heavy meromyosin (HMM) NTPase, the rates of NTP binding to myosin and actomyosin, NTP-mediated acto-HMM dissociation, and NTP hydrolysis by acto-HMM were made for comparison to the mechanical results. The data suggest a very similar mechanism of acto-HMM NTP hydrolysis. Whereas all NTPs studied support force production and stiffness that vary by a factor 2 or less, the unloaded shortening velocity (Vu) of muscle fibers varies by almost 10-fold. 2-Deoxy ATP (dATP) was unique in that Vu was 30% greater than with ATP. Parallel behavior was observed between Vf and the steady-state maximum actin-activated HMM ATPase rate. Further comparisons suggest that the variation in force correlates with the rate and equilibrium constant for NTP cleavage; the variations in Vu or Vf are related to the rate of cross-bridge dissociation caused by NTP binding or to the rate(s) of product release.
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PMID:ATP analogs and muscle contraction: mechanics and kinetics of nucleoside triphosphate binding and hydrolysis. 963 59

In the maturation of the Escherichia coli antibiotic Microcin B17, the product of the mcbA gene is modified posttranslationally by the multimeric Microcin synthetase complex (composed of McbB, C, and D) to cyclize four Cys and four Ser residues to four thiazoles and four oxazoles, respectively. The purified synthetase shows an absolute requirement for ATP or GTP in peptide substrate heterocyclization, with GTP one-third as effective as ATP in initial rate studies. The ATPase/GTPase activity of the synthetase complex is conditional in that ADP or GDP formation requires the presence of substrate; noncyclizable versions of McbA bind to synthetase, but do not induce the NTPase activity. The stoichiometry of ATP hydrolysis and heterocycle formation is 5:1 for a substrate that contains two potential sites of modification. However, at high substrate concentrations (>50Km) heterocycle formation is inhibited, while ATPase activity occurs undiminished, consistent with uncoupling of NTP hydrolysis and heterocycle formation at high substrate concentrations. Sequence homology reveals that the McbD subunit has motifs reminiscent of the Walker B box in ATP utilizing enzymes and of motifs found in small G protein GTPases. Mutagenesis of three aspartates to alanine in these motifs (D132, D147, and D199) reduced Microcin B17 production in vivo and heterocycle formation in vitro, suggesting that the 45 kDa McbD has a regulated ATPase/GTPase domain in its N-terminal region necessary for peptide heterocyclization.
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PMID:ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin B17. 974 32

The intact virion of bluetongue virus comprises ten segments of dsRNA enclosed in two concentric protein capsids. The core, which is transcriptionally active, includes three minor proteins (VP1, VP4 and VP6) which are considered to be the candidates for the core-associated enzymes that transcribe and modify full-length mRNA copies for each of the ten genome segments. Using purified recombinant VP4 protein and core-like particles containing VP4, in this report it is demonstrated that VP4 has nucleoside triphosphatase (NTPase) activity. VP4 is a nonspecific NTPase that hydrolyses four types of ribonucleoside triphosphate (NTP) to the corresponding nucleoside diphosphate. The substrate preference was GTP>ATP>UTP>CTP. NTP hydrolysis by VP4 was maximal when the Mg2+ or Ca2+ ion concentrations were 4 mM or 6 mM, respectively. The presence of single-stranded polynucleotides poly(A), poly(U) and poly(C) had little effect on the NTPase activity. Although the enzyme exhibited a broad temperature optimum around 40 degrees C, the pH optimum was sharp, between pH 7.5 and 8. The Km and Vmax of ATP hydrolysis were calculated to be 0.25+/-0.05 microM ATP and 55+/-4 pmol ATP hydrolysed min(-1) microg(-1), respectively. The Km was affected by the addition of poly(A) to only a small extent in contrast to the Vmax, which was increased by at least twofold.
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PMID:Bluetongue virus core protein VP4 has nucleoside triphosphate phosphohydrolase activity. 978 54

Although membrane associated enzymes such as ATPase, alkaline phosphatase, and NTP pyrophosphohydrolase in matrix vesicles (MVs) may underlie the mechanisms of ATP-promoted calcification, prior to the current investigation, the role of the MV membrane in calcification had not been addressed. In this study, various perturbations were introduced to the MV membrane in in vitro calcification systems to determine ideal conditions for ATP-initiated calcification by MVs isolated from rachitic rat epiphyseal cartilage. Membrane integrity appears to be required, since the rupture of the vesicular membrane by vigorously mixing with 10% butanol abolished calcification. In contrast, a mild treatment of MVs with low concentrations (e.g., 0.01%, which is much below the critical concentration for micelle formation) of either neutral Triton X-100 or anionic deoxycholate stimulated calcification by >2-fold, without inducing obvious changes in vesicular appearance. Fourier transform infrared spectroscopic studies were done to identify the mineral phase formed in these experiments. For the first time, rachitic MVs were shown to induce the formation of a calcium pyrophosphate dihydrate-like phase after their exposure to calcifying medium with 1 mM ATP. The integration of spectral areas indicated that calcification was enhanced by Triton X-100. The detergent effect was reversible and appeared to be not mediated through activation of ATPase, alkaline phosphatase, or ATP pyrophosphohydrolase. In contrast to neutral Triton X-100 and anionic deoxycholate, cationic cetyltrimethylammonium bromide inhibited both ATPase activity (I50=10 microM) and ATP-initiated calcification. These observations suggest that membrane perturbations can affect calcification and that the presence of NTP-pyrophosphohydrolase in MVs may play a role in the deposition of CaPPi in rachitic cartilage.
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PMID:Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles. 988 89

Advanced mineralization can cause brittleness of aortic walls with decreased elasticity thereby causing the wall to rupture. Although the precise mechanisms of dystrophic calcification remain unknown, morphological evidence reveals the presence of mineral-associated vesicles in the lesions and defective bioprosthetic valves. In an attempt to demonstrate the calcifiability of the vesicles, small segments of human atherosclerotic aortas with calcified lesions were removed at autopsy and then digested in a crude collagenase solution to release vesicles. A differential centrifugation was then used to isolate calcifiable vesicles, which was precipitated at 300,000 x g for 20 min. An exposure of the vesicles to a calcifying medium containing physiologic levels of Ca2+, Pi, and 1 mM ATP caused Ca deposition in a vesicle protein-concentration dependent manner. The calcifiability of the vesicles was further demonstrated by electron microscopy. Fourier transform spectroscopic analysis of the deposited mineral revealed the presence of a hydroxyapatite phase, closely resembling the native form of mineral in atherosclerotic plaques. In addition, calcifiable vesicles were enriched in ATP-hydrolyzing enzymes including Mg2+ or Ca2+-ATPase and NTP pyrophosphohydrolase that may be involved in normal and pathological calcification. Triton X-100 at 0.01% abolished 80% of both ATPase activity and ATP-initiated calcification. A comparison of vesicles isolated from non-atherosclerotic and atherosclerotic aortas indicated that atherosclerotic vesicles tended to have higher calcifiability. These observations suggest that the calcifiable vesicles play a part in dystrophic calcification of aortas in atherosclerosis.
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PMID:Isolation of calcifiable vesicles from human atherosclerotic aortas. 1021 64

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are activated by the Ino2p/Ino4p transcription factor that binds to ICRE promoter motifs and mediates maximal gene expression in the absence of inositol. We identified the ino80 mutation causing inositol auxotrophy as a result of a defect in ICRE-dependent gene activation. The product of the corresponding wild-type gene INO80 (= YGL150C) shows significant similarity to the Snf2p family of DNA-dependent ATPases. Nevertheless, SNF2 in increased gene dosage did not suppress ino80 mutant phenotypes. Mutation of the Ino80p lysine residue corresponding to the NTP binding site of Snf2p led to a non-functional protein. In ino80 null mutants, gene activation mediated by an ICRE decreased to 16% of the wild-type level. Maximal expression of PHO5, GAL1, CYC1 and ICL1 was also significantly reduced. Thus, Ino80p affects several transcription factors involved in unrelated pathways. As demonstrated by gel filtration, Ino80p is part of a high-molecular-weight complex of more than 1 MDa. Similar to what was found for Snf2p, the Ino80p-containing complex may influence the transcriptional level of several unrelated structural genes by functioning as an ATPase that possibly acts on chromatin.
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PMID:The product of the SNF2/SWI2 paralogue INO80 of Saccharomyces cerevisiae required for efficient expression of various yeast structural genes is part of a high-molecular-weight protein complex. 1036 Dec 78


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