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)

Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites.
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PMID:Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase. 243 24

Levels of calmodulin and activities of calcium and magnesium ATPases were determined in renal tissues of age-matched male rats after increasing durations of the following conditions: untreated streptozotocin (STZ) induced diabetes; STZ-diabetic rats which received daily insulin (NPH) treatment beginning 24 hours after STZ administration; and STZ-diabetic rats which began receiving NPH after having endured untreated diabetes for 1, 2, and 3 weeks. Results were compared with those of age-matched control animals. Calmodulin levels were the same in renal tissues from all groups of rats as were magnesium-ATPase activities. Calcium-ATPase activities were significantly higher than control activities after 2 weeks of untreated STZ-diabetes and in tissues from all rats that received insulin treatment which was instituted after 1, 2 and 3 weeks of STZ-diabetes. Calcium ATPase activities of tissues from rats which received insulin treatment 24 hrs after STZ injections were no different from control values. The data indicate that prolonged deficiency of insulin results in insufficient passive transport of calcium from urine into tubular cells. Thus, the increased activity of calcium-ATPase on the contraluminal membrane of kidney cells represents a futile attempt to conserve calcium.
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PMID:Calmodulin levels and divalent cation pump activities in kidneys of streptozotocin-diabetic rats. 613 28

We have carried out a detailed analysis of viral mRNAs and proteins produced in cultured cells infected with a temperature-sensitive vaccinia virus mutant (ts36) containing a modified nucleoside triphosphate phosphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a recombinant virus (ts36LUC) which expresses the luciferase marker, we showed in seven different cell lines that early expression of the receptor gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temperature. The steady-state levels of different early viral polypeptides were also severely reduced. Analysis of steady-state mRNA levels for two early genes (DNA polymerase and D5) showed that inhibition of early polypeptide synthesis correlated with a reduction in the levels of mRNA accumulated at the nonpermissive temperature. Analysis of steady-state levels of late viral polypeptides and of mRNAs indicated that NPH-I regulation of intermediate and late gene expression is direct and not simply a consequence of its role in inhibiting early gene expression. Characterization of a rescued virus (R36) demonstrated that the temperature-sensitive phenotype of ts36 is due solely to the point mutation in the NPH-I gene. The mutant phenotype is not due to reduced levels of NPH-I present in ts36 virions or to the differential stability of this enzyme in cells infected at the nonpermissive temperature but to inhibition of normal enzymatic activity for this protein. Measurement of viral transcriptional activity in permeabilized purified virions demonstrated that NPH-I is required for normal rates of transcription in vaccinia virus. Our findings show ts36 to be a strongly defective early mutant of vaccinia virus and prove that NPH-I plays a key role in the control of early and late virus gene expression, possibly by way of an auxiliary function which regulates mRNA transcription during the virus growth cycle.
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PMID:Vaccinia virus nucleoside triphosphate phosphohydrolase I controls early and late gene expression by regulating the rate of transcription. 823 Apr 76

Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent.
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PMID:The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding. 862 91

Temperature-sensitive mutations (ts10, ts18, and ts39) of the vaccinia virus RNA helicase nucleoside triphosphate phosphohydrolase II (NPH-II) result in the production of noninfectious progeny virions at the restrictive temperature. The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of Western blot (immunoblot) analysis indicate that these particles lack NPH-II, whereas other enzymatic components of the virus core are present. These components include the following: DNA-dependent RNA polymerase subunits rpo147, rpo132, rpo94, rpo35, rpo30, rpo22, and rpo18; early transcription initiation factor subunits A8 and D6; mRNA capping enzyme subunits D1 and D12; RNA cap 2'-O-methyltransferase; A18 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase. Although RNA polymerase is encapsidated by the mutant viruses, mRNA synthesis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporation into acid-insoluble material. Moreover, the transcripts synthesized by the mutant particles are longer than normal and remain virion associated. Transcription initiation by mutant virions occurs accurately at an endogenous genomic promoter, albeit at reduced levels (1 to 7%) compared with that of wild-type virions. In contrast, extracts of the mutant virions catalyze the wild-type level of transcription from an exogenous template containing an early promoter. We conclude that NPH-II is required for early mRNA synthesis uniquely in the context of the virus particle. Possible roles in transcription termination and RNA transport are discussed.
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PMID:Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription. 897 Sep 79

Vaccinia virus RNA polymerase terminates transcription in response to a specific signal UUUUUNU in the nascent RNA. Transduction of this signal to the elongating polymerase requires a trans-acting viral termination factor (VTF/capping enzyme), and is coupled to the hydrolysis of ATP. Recent studies suggest that ATP hydrolysis is catalyzed by a novel termination protein (factor X), which is tightly associated with the elongation complex. Here, we identify factor X as NPH-I (nucleoside triphosphate phosphohydrolase-I), a virus-encoded DNA-dependent ATPase of the DExH-box family. We report that NPH-I serves two roles in transcription (1) it acts in concert with VTF/CE to catalyze release of UUUUUNU-containing nascent RNA from the elongation complex, and (2) it acts by itself as a polymerase elongation factor to facilitate readthrough of intrinsic pause sites. A mutation (K61A) in the GxGKT motif of NPH-I abolishes ATP hydrolysis and eliminates the termination and elongation factor activities. Related DExH proteins may have similar roles at postinitiation steps during cellular mRNA synthesis.
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PMID:Vaccinia NPH-I, a DExH-box ATPase, is the energy coupling factor for mRNA transcription termination. 947 22

Vaccinia virus NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by six shared sequence motifs. The contributions of conserved amino acids in motifs I (TGVGKTSQ), Ia (PRI), II (DExHE), and III (TAT) to enzyme activity were assessed by alanine scanning. NPH-II-Ala proteins were expressed in baculovirus-infected Sf9 cells, purified, and characterized with respect to their RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Alanine substitutions at Lys-191 and Thr-192 (motif I), Arg-229 (motif Ia), and Glu-300 (motif II) caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. In contrast, alanine mutations at His-299 (motif II) and at Thr-326 and Thr-328 (motif III) elicited defects in RNA unwinding but spared the ATPase. None of the mutations analyzed affected the binding of NPH-II to RNA. These findings, together with previous mutational studies, indicate that NPH-II motifs I, Ia, II, and VI (QRxGRxGRxxxG) are essential for nucleoside triphosphate (NTP) hydrolysis, whereas motif III and the His moiety of the DExH-box serve to couple the NTPase and helicase activities. Wild-type and mutant NPH-II-Ala genes were tested for the ability to rescue temperature-sensitive nph2-ts viruses. NPH-II mutations that inactivated the phosphohydrolase in vitro were lethal in vivo, as judged by the failure to recover rescued viruses containing the Ala substitution. The NTPase activity was necessary, but not sufficient, to sustain virus replication, insofar as mutants for which NTPase was uncoupled from unwinding (H299A, T326A, and T328A) were also lethal. We conclude that the phosphohydrolase and helicase activities of NPH-II are essential for virus replication.
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PMID:The nucleoside triphosphatase and helicase activities of vaccinia virus NPH-II are essential for virus replication. 957 37

Deng and Shuman (J. Biol Chem. 271, 29386 (1996)) reported that an ATPase different from the known viral termination factor, VTF, is required for vaccinia virus early gene transcription termination. Properties of this ATPase were similar to those of a known vaccinia virus enzyme, nucleoside triphosphate phosphohydrolase I (NPH I) the product of gene D11L. Transcription-competent cell-free extracts were prepared from A549 cells infected with wild-type or mutant vaccinia virus harboring ts mutations in gene D11L. These extracts were employed to investigate the role of NPH I in early gene transcription termination. Extracts prepared under nonpermissive conditions from both wild-type virus and ts mutant virus-infected cells exhibited high levels of early and intermediate gene transcription activity but were incapable of supporting late gene transcription. ts mutant extract lacked signal-dependent early gene transcription termination activity, which was restored by the addition of either free NPH I or a GST-NPH I fusion protein. A comparison of the NPH I amino acid sequence to the protein databases revealed the presence of a set of sequences characteristic of nucleic acid helicase superfamily II members. A series of site-specific mutations in the helicase motifs and N-terminal and C-terminal deletion mutations were expressed as GST fusion proteins and their activities assessed. Of the mutations in helicase motifs I to VI, alteration of all but motif III reduced the ATPase activity. Removal of as few as 24 amino acids from the N-terminal end eliminated ATPase activity, while deletion of 68 C-terminal amino acids exhibited only a modest decrease in ATP hydrolysis. Larger C-terminal deletions eliminated ATPase activity. Each deletion mutation, and site-specific mutations other than the motif III mutation, failed to support transcription termination in vitro. Mutations in motifs I, II, V, and VI inhibit wild-type NPH I transcription termination activity. However, deletion of up to 68 amino acids from the C-terminal end eliminates this inhibitory property. This observation is particularly interesting since these C-terminal deletions retain both ATPase activity and single-stranded DNA binding activity. Their failure to inhibit transcription termination suggests that these C-terminal deletion mutations eliminate a site required for a function other than from DNA binding or ATP hydrolysis.
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PMID:Vaccinia virus nucleoside triphosphate phosphohydrolase I is an essential viral early gene transcription termination factor. 963 76

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) is a DNA-dependent ATPase that serves as a transcription termination factor during viral mRNA synthesis. NPH-I is a member of the DExH box family of nucleic acid-dependent nucleoside triphosphatases (NTPases), which is defined by the presence of several conserved sequence motifs. We have assessed the contributions of individual amino acids (underlined) in motifs I (GxGKT), II (DExHN), III (SAT), and VI (QxxGRxxR) to ATP hydrolysis by performing alanine scanning mutagenesis. Significant decrements in ATPase activity resulted from mutations at nine positions: Lys-61 and Thr-62 (motif I); Asp-141, Glu-142, His-144, and Asn-145 (motif II); and Gln-472, Arg-476, and Arg-479 (motif VI). Structure-function relationships at each of these positions were clarified by introducing conservative substitutions and by steady-state kinetic analysis of the mutant enzymes. Comparison of our findings for NPH-I with those of mutational studies of other DExH and DEAD box proteins underscores similarities as well as numerous disparities in structure-activity relationships. We conclude that the functions of the conserved amino acids of the NTPase motifs are context dependent.
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PMID:Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase of the DExH box family. 988 35

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) serves as the ATPase activity employed in early gene transcription termination [Deng, L., and Shuman, S. (1998) Genes Dev. 12, 538-546; Christen, L. M., et al. (1998) Virology 245, 360-371]. Since ATPase activity requires binding of single-stranded DNA, a full understanding of the mechanism of oligonucleotide activation is essential for the elucidation of its role in transcription termination. To initiate detailed structure-function studies of NPH I, we undertook combined kinetic and binding analyses of the interaction of linear oligonucleotides with NPH I. In the presence of single-stranded DNA, ATP exhibits complex saturation kinetics. The apparent Km for ATP is independent of DNA concentration, demonstrating that ssDNA binding alters the kcat for the reaction. Linear ssDNA oligonucleotides from 18 to 48 nucleotides in length stimulated activity in a saturatable fashion. As the oligonucleotide length increases, the Kact decreases and the Vmax increases. The increase in affinity is paralleled by an increase in the level of binding as measured by EMSA. The kinetic activation observed for 36-nucleotide ssDNA is dependent upon ATP concentration. At low ATP levels, sigmoidal saturation kinetics are observed, while at saturating ATP levels, near-hyperbolic kinetics are seen, suggesting that NPH I may adopt two conformational states. Linear oligonucleotides 18, 24, and 36 bases in length bind one, two, and three molecules of NPH I maximally, respectively, indicating that the NPH I binding site is no more than 12 bases in length. In contrast, single-stranded RNA does not stimulate ATPase activity, yet RNA binds as well as DNA of a similar length. Both RNA and DNA can be photo-cross-linked to NPH I by UV light. ssDNA and ssRNA cross-compete in UV photo-cross-linking to NPH I, indicating that both oligonucleotides share a common binding site. ssRNA prevents ssDNA activation of ATPase activity, confirming that both oligonucleotides bind to the kinetically important oligonucleotide activation site on NPH I. ssDNA inhibits transcription termination in vitro. Inhibition is overcome by adding NPH I, demonstrating that oligonucleotide inhibition is mediated through NPH I.
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PMID:Interaction of the vaccinia virus nucleoside triphosphate phosphohydrolase I with linear oligonucleotides. 1038 52


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