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)

We previously described the [(3)H]cGMP-binding characteristics of a CHAPS-solubilized protein that we proposed to be a cGMP transporter. We now report the ATPase activity of the membrane-bound, solubilized and reconstituted form of a cGMP transporter. The membrane-bound protein of unsealed ghosts had a linear ATPase activity over a 120 min incubation period with optimal activity of about 400 pmol/mg/min. The apparent K(m) and V(max) for ATP were about 0.5 mM and 300 pmol/mg/min, respectively. When solubilized with CHAPS the specific activity of the protein was reduced to about 70 pmol/mg/min. Reconstitution of the CHAPS preparation into phospholipid bilayer using rapid detergent removal by Extracti-gel column resulted in proteoliposomes which had ATPase activity similar to that found in the erythrocyte membranes. The proteoliposomes displayed a linear ATP-dependent uptake of [(3)H]cGMP with an apparent K(m) value of 1. 0 microM. This low K(m)-uptake of [(3)H]cGMP in proteoliposomes was not affected by 10 microM of AMP, cAMP and GMP, but was completely abolished in the presence of the non-hydrolyzable ATP analogue, ATP-gamma-S. Some ATPase activation was also observed in the presence of 2 microM cAMP, but it is unclear whether this activity was coupled to the cGMP transporter. Our results show that the membrane protein responsible for cGMP transport has an ATPase activity and transports the cyclic nucleotide in the presence of ATP.
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PMID:ATPase activity and transport by a cGMP transporter in human erythrocyte ghosts and proteoliposome-reconstituted membrane extracts. 1111 55

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the beta-gamma phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast SCHIZOSACCHAROMYCES: pombe. Pct1p hydrolyzes the gamma phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and P(i) in the presence of manganese or cobalt (K(m) = 19 microM ATP; k(cat) = 67 s(-1)). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I(0.5) = 1 mM), pyrophosphate (I(0.5) = 0.4 mM) and tripolyphosphate (I(0.5) = 30 microM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.
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PMID:Characterization of Schizosaccharomyces pombe RNA triphosphatase. 1113 8

Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.
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PMID:RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1. 1116 Jun 72

A gene encoding a putative GTPase containing two tandemly repeated GTP-binding domains from a hyperthermophilic bacterium, Thermotoga maritima, was cloned and expressed in Escherichia coli. The gene (TM1446) termed der is highly conserved in Eubacteria including E. coli. The purified der product (Tm-Der) has GTPase activity but no ATPase activity. GTP, GDP, and dGTP but not GMP, ATP, CTP, and UTP compete for GTP binding to Tm-Der. An optimal condition for the GTPase assay was determined to be pH 7.5 in 400 mm KCl and 5 mm MgCl(2) at 70 degrees C, where K(m), V(max), and k(cat) values were determined to be 110 microm, 3.46 microm/min, and 0.87 min(-1), respectively. A der deletion strain of E. coli was constructed by replacing the der gene (originally annotated yfgK) with a kanamycin resistance gene. The deletion strain was found to form colonies only if the cells harbored a plasmid containing der, indicating that der is essential for E. coli growth.
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PMID:An essential GTPase, der, containing double GTP-binding domains from Escherichia coli and Thermotoga maritima. 1138 44

The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5'-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid-specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements.
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PMID:Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA-triphosphatase activities. 1142 64

Unique transcripts for cytochrome b, ATPase subunits 6 and 9, cytochrome oxidase subunits 2 and 3 and S and L rRNA have been mapped by the S1 protection technique to the circular 19-kbp mitochondrial DNA (mtDNA) of the yeast Torulopsis glabrata. In contrast, a number of transcripts have been detected for the mosaic cytochrome oxidase subunit 1 gene with the largest being approximately 5000 nucleotides and the mature message having a length of 1760 nucleotides. Despite the presence in T. glabrata mtDNA of a sequence that hybridizes to the variant 1 gene of Saccharomyces cerevisiae mtDNA we have not detected a transcript of this region. Neither have we detected co-transcripts of adjacent genes in RNA from either glucose-repressed or derepressed cells. However, by comparison of RNA species from the two growth conditions, we have found that the ATPase subunit 6 transcript is lower in amount relative to other species in preparations from glucose-repressed cells. This information, together with the observation of separate transcripts and the knowledge that there are several species of mitochondrial RNA which can be capped by the guanylyl transferase catalysed addition of GMP, suggests that each of the genes investigated in the present study is separately transcribed.
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PMID:Map location of transcripts from Torulopsis glabrata mitochondrial DNA. 1189 97

Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H(+)-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5'-O-2-thiodiphosphate, GTP, or 5'-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.
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PMID:Guanine derivatives modulate L-glutamate uptake into rat brain synaptic vesicles. 1468 7

In the presence of NMDA receptor open-channel blockers [Mg(2+); (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801); 1-amino-3,5-dimethyladamantane (memantine)] and TTX, high concentrations (30-100 microm) of either 5-hydroxytryptamine (5-HT) or alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) significantly potentiated NMDA-induced depolarizations of frog spinal cord motoneurones. Potentiation was blocked by LY-53,857 (10-30 microm), SB 206553 (10 microm), and SB 204741 (30 microm), but not by spiroxatrine (10 microm), WAY 100,635 (1-30 microm), ketanserin (10 microm), RS 102221 (10 microm), or RS 39604 (10-20 microm). Therefore, alpha-Me-5-HT's facilitatory effects appear to involve 5-HT(2B) receptors. These effects were G-protein dependent as they were prevented by prior treatment with guanylyl-5'-imidodiphosphate (GMP-PNP, 100 microm) and H-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH(2) (GP antagonist 2A, 3-6 microm), but not by pertussis toxin (PTX, 3-6 ng ml(-1), 48 h preincubation). This potentiation was not reduced by protein kinase C inhibition with staurosporine (2.0 microm), U73122 (10 microm) or N-(2-aminoethyl)-5-isoquinolinesulfonamide HCl (H9) (77 microm) or by intracellular Ca(2+) depletion with thapsigargin (0.1 microm) (which inhibits Ca(2+)/ATPase). Exposure of the spinal cord to the L-type Ca(2+) channel blockers nifedipine (10 microm), KN-62 (5 microm) or gallopamil (100 microm) eliminated alpha-Me-5-HT's effects. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) (100 microm) diminished the potentiation. However, the calcium/calmodulin-dependent protein kinase II (CaM Kinase II) blocker KN-93 (10 microm) did not block the 5-HT enhancement of the NMDA responses. In summary, activation of 5-HT(2B) receptors by alpha-Me-5-HT facilitates NMDA-depolarizations of frog motoneurones via a G-protein, a rise in [Ca(2+)](i) from the entry of extracellular Ca(2+) through L-type Ca(2+) channels, the binding of Ca(2+) to calmodulin and a lessening of the Mg(2+) -produced open-channel block of the NMDA receptor.
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PMID:Mechanisms intrinsic to 5-HT2B receptor-induced potentiation of NMDA receptor responses in frog motoneurones. 1533 59

Banna virus (BAV) particles contain seven structural proteins: VP4 and VP9 form an outer-capsid layer, whilst the virus core contains three major proteins (VP2, VP8 and VP10) and two minor proteins (VP1 and VP3). Sequence analysis showed that VP3 contains motifs [Kx(I/V/L)S] and (Hx(n)H) that have previously been identified in the guanylyltransferases of other reoviruses. Incubation of purified BAV-Ch core particles with [alpha-32P]GTP resulted in exclusive covalent labelling of VP3, demonstrating autoguanylation activity (which is considered indicative of guanylyltransferase activity). Recombinant VP3 prepared in a cell-free expression system was also guanylated under similar reaction conditions, and products were synthesized (in the presence of non-radiolabelled GDP) that co-migrated with GMP, GDP and GpppG during TLC. This reaction, which required magnesium ions for optimum activity, demonstrates that VP3 possesses nucleoside triphosphatase (GTPase) activity and is the BAV guanylyltransferase (RNA 'capping' enzyme).
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PMID:Identification and functional analysis of VP3, the guanylyltransferase of Banna virus (genus Seadornavirus, family Reoviridae). 1578 8

Uptake of neurotransmitters into synaptic vesicles is driven by an electrochemical gradient generated by a vacuolar-type proton pump ATPase. This uptake implies a key role for synaptic vesicles in the regulation of neurotransmitter systems. Guanine nucleoside and nucleotides are involved in the inhibition of glutamate-induced cellular responses via an extracellular action and diverse trophic, proliferative, and modulatory effects of guanine nucleotides on neural cells have been shown. Here, we characterized the uptake of GTP into synaptic vesicles isolated from whole rat brain, by using a tritiated poorly-hydrolyzable GTP analog, 5'-guanylylimidodiphosphate ([3H]GppNHp). Uptake of GTP into synaptic vesicles is saturable, time- and temperature-dependent, and relies on a proton-eletrochemical gradient. However, [3H]GMP and [3H]GDP radioactive labeling in synaptic vesicles is not dependent on temperature and vesicular ATPase activity, which indicates that these nucleotides only bind to and are not taken up into synaptic vesicles. GTP is taken up by the same eletrochemical gradient-dependent transport system, as are neurotransmitters storage, which indicates that this guanine nucleotide may also function as a neurotransmitter.
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PMID:GTP uptake into rat brain synaptic vesicles. 1640 24


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