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)

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.
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PMID:Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon. 1518 86

Lesch-Nyhan disease (LND) is a rare disorder caused by a defect of an enzyme in the purine salvage pathway, hypoxanthine phosphoribosyl transferase (HPRT). It is still unknown how the metabolic defect translates into the complex neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in purine and pyrimidine nucleotide content in HPRT-deficient cells. We hypothesized that altered nucleotide concentrations in HPRT deficiency change G-protein-mediated signal transduction. Therefore, our original study aim was to examine the high-affinity GTPase activity of G-proteins in membranes from primary human skin and immortalized mouse skin fibroblasts, rat B103 neuroblastoma cells and mouse Neuro-2a neuroblastoma cells. Unexpectedly, in membranes from human fibroblasts, B103- and Neuro-2a cells, V(max) of low-affinity nucleoside 5'-triphosphatase (NTPase) activities was decreased up to 7-fold in HPRT deficiency. In contrast, in membranes from mouse fibroblasts, HPRT deficiency increased NTPase activity up to 4-fold. The various systems analyzed differed from each other in terms of K(m) values for NTPs, absolute V(max) values and K(i) values for nucleoside 5'-[beta,gamma-imido]triphosphates. Our data show that altered membrane NTPase activity is a biochemical hallmark of HPRT deficiency, but species and cell-type differences have to be considered. Thus, future studies on biochemical changes in LND should be conducted in parallel in several HPRT-deficient systems.
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PMID:Altered membrane NTPase activity in Lesch-Nyhan disease fibroblasts: comparison with HPRT knockout mice and HPRT-deficient cell lines. 1593 74

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and reduction is widely used to evaluate cell proliferation and viability. MTT is taken up by the cells through endocytosis. We find that ouabain (1-200 nM) inhibits MTT reduction in human umbilical vein endothelial cells (HUVEC) without affecting cell viability. Ouabain does not inhibit MTT reduction when cell lysates substituted for the intact cells. Disruption of caveolae by cholesterol depletion, completely prevents the effect of ouabain. Treatment of HUVEC with Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine partially abrogates the inhibitory effect of ouabain. The data suggest that ouabain interaction with caveolar Na/K-ATPase inhibits MTT endocytosis through the activation of signaling proteins such as Src kinase.
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PMID:Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) endocytosis by ouabain in human endothelial cells. 1664 3

Three members of the Nudix (nucleoside diphosphate X) hydrolase superfamily have been cloned from Escherichia coli MG1655 and expressed. The proteins have been purified and identified as enzymes active on nucleoside diphosphate derivatives with the following specificities. Orf141 (yfaO) is a nucleoside triphosphatase preferring pyrimidine deoxynucleoside triphosphates. Orf153 (ymfB) is a nonspecific nucleoside tri- and diphosphatase and atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. Orf191 (yffH) is a highly active GDP-mannose pyrophosphatase. All three enzymes require a divalent cation for activity and are optimally active at alkaline pH, characteristic of the Nudix hydrolase superfamily. The question of whether or not Orf1.9 (wcaH) is a bona fide member of the Nudix hydrolase superfamily is discussed.
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PMID:Three new Nudix hydrolases from Escherichia coli. 1676 26

A model for the origin of biochemistry at an alkaline hydrothermal vent has been developed that focuses on the acetyl-CoA (Wood-Ljungdahl) pathway of CO2 fixation and central intermediary metabolism leading to the synthesis of the constituents of purines and pyrimidines. The idea that acetogenesis and methanogenesis were the ancestral forms of energy metabolism among the first free-living eubacteria and archaebacteria, respectively, stands in the foreground. The synthesis of formyl pterins, which are essential intermediates of the Wood-Ljungdahl pathway and purine biosynthesis, is found to confront early metabolic systems with steep bioenergetic demands that would appear to link some, but not all, steps of CO2 reduction to geochemical processes in or on the Earth's crust. Inorganically catalysed prebiotic analogues of the core biochemical reactions involved in pterin-dependent methyl synthesis of the modern acetyl-CoA pathway are considered. The following compounds appear as probable candidates for central involvement in prebiotic chemistry: metal sulphides, formate, carbon monoxide, methyl sulphide, acetate, formyl phosphate, carboxy phosphate, carbamate, carbamoyl phosphate, acetyl thioesters, acetyl phosphate, possibly carbonyl sulphide and eventually pterins. Carbon might have entered early metabolism via reactions hardly different from those in the modern Wood-Ljungdahl pathway, the pyruvate synthase reaction and the incomplete reverse citric acid cycle. The key energy-rich intermediates were perhaps acetyl thioesters, with acetyl phosphate possibly serving as the universal metabolic energy currency prior to the origin of genes. Nitrogen might have entered metabolism as geochemical NH3 via two routes: the synthesis of carbamoyl phosphate and reductive transaminations of alpha-keto acids. Together with intermediates of methyl synthesis, these two routes of nitrogen assimilation would directly supply all intermediates of modern purine and pyrimidine biosynthesis. Thermodynamic considerations related to formyl pterin synthesis suggest that the ability to harness a naturally pre-existing proton gradient at the vent-ocean interface via an ATPase is older than the ability to generate a proton gradient with chemistry that is specified by genes.
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PMID:On the origin of biochemistry at an alkaline hydrothermal vent. 1725 2

5-Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6-hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1-overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.
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PMID:Overexpression of HAM1 gene detoxifies 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae. 1789 88

A new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis subsp. lactis biovar diacetylactis strain DB0410. This plasmid is responsible for the sensitivity of DB0410 to the toxic pyrimidine analogue 5-fluoroorotate. The complete nucleotide sequence has been determined and amounts to 16 404 bp. Of 15 ORFs encountered, three were found to be insertion sequence (IS) elements, identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their C-terminal region. Homologues for four ORFs were found in the IL1403 sequence: the copB gene, coding for a copper-potassium-transporting ATPase B, and the ysbA, ysbB and ysbC genes. The structural organization of the pDBORO replication region is highly similar to other theta-replicating plasmids in both the cis- (repA) and trans-acting (repB and orfX) sequences. By plasmid deletion analysis and molecular cloning, a single locus on pDBORO was found to confer sensitivity to 5-fluoroorotate. It was identified as ysbC, but renamed oroP in order to reflect its function. The oroP gene was found to be essential for the utilization of orotate as the sole pyrimidine source in a strain deficient in pyrimidine de novo synthesis. The amino acid sequence encoded by the ORF showed the characteristic features of a membrane protein. Therefore, oroP most probably encodes an orotate transporter. Surprisingly, homologues of oroP could be identified in the genomes of both L. lactis MG1363 and L. lactis IL1403 despite the fact that these strains were unable to significantly utilize orotate. Cloning of oroP in Escherichia coli and Bacillus subtilis showed that the orotate transport phenotype could be transformed to both organisms. The findings presented indicate that oroP can be used as a powerful, food-grade selection/counterselection marker in many different organisms.
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PMID:The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker. 1797 72

The interaction between 2',3'-cyclic nucleotide 3'-phosphodiesterase and guanine/adenine nucleotides was investigated. The binding of purine nucleotides to 2',3'-cyclic nucleotide 3'-phosphodiesterase was revealed by both direct and indirect methods. In fact, surface plasmon resonance experiments, triphosphatase activity measurements, and fluorescence experiments revealed that 2',3'-cyclic nucleotide 3'-phosphodiesterase binds purine nucleotide triphosphates with an affinity higher than that displayed for diphosphates; on the contrary, the affinity for both purine monophosphates and pyrimidine nucleotides was negligible. An interpretation of biological experimental data was achieved by a building of 2',3'-cyclic nucleotide 3'-phosphodiesterase N-terminal molecular model. The structural elements responsible for nucleotide binding were identified and potential complexes between the N-terminal domain of CNP-ase and nucleotide were analyzed by docking simulations. Therefore, our findings suggest new functional and structural property of the N-terminal domain of CNPase.
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PMID:The N-terminal domain of 2',3'-cyclic nucleotide 3'-phosphodiesterase harbors a GTP/ATP binding site. 1798 4

Similar to the assembly of other dsDNA viruses, bacterial virus phi29 uses a motor to translocate its DNA into a procapsid, with the aid of protein gp16 that binds to pRNA 5'/3' helical region. To investigate the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was evaluated as a function of DNA structure (ss- or ds-stranded) or chemistry (purine or pyrimidine). The k(cat) and K(m) in the absence of DNA was 0.016 s(-1) and 351.0 microM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of DNA or pRNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine)>dsDNA>poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involved in the contact with gp16 during DNA packaging.
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PMID:Strand and nucleotide-dependent ATPase activity of gp16 of bacterial virus phi29 DNA packaging motor. 1870 Nov 24

The tegumental membrane of Taenia crassiceps cysticerci contains an ATP-diphosphohydrolase (EC 3.6.1.5) which hydrolyzes purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates at an optimum pH of 8.5. It is Mg(2+)-dependent and insensitive to classical ATPase and phosphatase inhibitors. In solubilized tegumental membrane the Km values varied from 220 to 480 microM and the V(max) from 370 to 748 nmol of Pi release/mg/min for nucleoside triphosphates (ATP, GTP, CTP, UTP, and TTP); for nucleoside diphosphates (ADP, GDP, CDP, and UDP) the Km values were from 260 to 450 microM and the V(max) from 628 to 1134 nmol of Pi release/mg/min. An antibody specific to CD39 shows cross-reactivity with T. crassiceps ATP-diphosphohydrolase, revealing a single protein of approximately 80 kDa. Incubation of ATP-diphosphohydrolase with FSBA inhibited ATPase and ADPase activities by 85-90%. Immunoblot analyses, the competition plot, similar inhibition by free nucleotides, the lack of effect of Mg(2+) at high concentrations, and the inactivation by FSBA of ATPase and ADPase activity strongly suggest that a single enzyme catalyzes the hydrolysis of all these nucleotides. The mechanism of ATP hydrolysis shows that ATP-diphosphohydrolase releases ADP during the catalytic cycle. Incubation of intact cysticerci with FSBA caused 70-80% inhibition of ATPase and ADPase activities, indicating that the active site of the ATP-diphosphohydrolase is oriented to the external surface of the tegument of T. crassiceps. The importance of this enzyme in the parasite-host relationship is discussed.
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PMID:5'-p-Fluorosulfonyl benzoyl adenosine inhibits an ecto-ATP-diphosphohydrolase in the tegument surface of Taenia crassiceps cysticerci. 1876 60


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