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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)

1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
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PMID:Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase. 15 44

The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed.
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PMID:Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3'-deoxyadenosine (cordycepin). 22 73

The human T-lymphoblastoid cell line CCRF-CEM, pre-treated with 2'-deoxycoformycin, was used as a model for adenosine deaminase deficiency to investigate how 2'-deoxyadenosine exerts its cytotoxic effects. Incubation of these cells with 1 microM or 5 microM deoxyadenosine for 24 and 48 h caused an increase of up to 50% in their modal cell volume as measured by a Coulter Size Distribution Analyzer and this increase in cell volume was accompanied by an increase in their fragility and deformability. The swelling of cells was concomitant with the phosphorylation of deoxyadenosine and its intracellular accumulation as dATP. There was no evidence of osmotic imbalance or of inhibition of the Na+/K(+)-dependent ATPase activity as the intracellular concentrations (and the intracellular:extracellular ratios) of Na+, K+ and Ca2+ were essentially unchanged. Cytochalasin B (20 microM) also caused lymphoblasts to swell over a 6-h period and its effect on cell size was similar to that of either 1 microM or 5 microM deoxyadenosine over 24 or 48 h. Longer time-courses of incubation with cytochalasin B caused severe toxicity leading to the death and lysis of a significant proportion of the cells. Other drugs, such as colchicine, vincristine and vinblastine that are known to affect various components of the cytoskeleton also caused swelling of cells in a concentration- and time-dependent manner but there was no evidence that these effects were additive or synergistic with those of deoxyadenosine. Inhibition of DNA synthesis, either directly by aphidicolin or indirectly by hydroxyurea, was less cytotoxic than the effect caused by deoxyadenosine. We conclude that one of the toxic effects resulting from the excessive phosphorylation of deoxyadenosine and its accumulation as dATP in human T-lymphoblasts is not dependent on inhibition of DNA synthesis but may be caused by the disruption of the cytoskeleton in these cells.
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PMID:Deoxyadenosine toxicity in an adenosine deaminase-inhibited human CCRF-CEM T-lymphoblastoid cell line causes cell swelling. 146 67

The interactions of the 70-kDa heat-shock proteins (hsp70s) with their protein substrates appear to be regulated by bound nucleotide. Previous work has shown that the nucleotide binding site of the bovine brain uncoating ATPase, a constitutive member of the hsp70 family, crystallographically resembles the nucleotide binding site of actin and, like actin, the uncoating ATPase has a strongly bound ADP which cannot be removed by dialysis or treatment with ethylenediaminetetraacetic acid (EDTA). This suggests that, like the bound nucleotide of actin, it may be required for the enzyme to retain its native structure. In this study, the strongly bound ADP was removed by first replacing it with 5'-adenylyl imidodiphosphate (AMP-PNP) and then removing the bound AMP-PNP by dialysis. Following this treatment, more than 95% of the uncoating ATPase becomes nucleotide-free. The nucleotide-free uncoating ATPase retains its ability to bind and hydrolyze ATP and to uncoat clathrin-coated vesicles, even after 10 days of storage at 4 degrees C. Therefore, in contrast to actin, the bound nucleotide of the uncoating ATPase is not required to prevent denaturation of the enzyme. Using nucleotide-free uncoating ATPase, we were able to accurately measure the dissociation constants of ATP, ADP, and the nucleotide analogues AMP-PNP and 2'-deoxyadenosine 5'-triphosphate (dATP). The dissociation constants of both ATP and ADP are about 10(-8) M, more than 1-2 orders of magnitude stronger than previously reported, while AMP-PNP and dATP bind 2-3 orders of magnitude more weakly than ATP.
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PMID:Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues. 811 62

2'-Deoxyadenosine 5'-triphosphate, 3'-deoxyadenosine 5'-triphosphate, and 3'-amino-3'-deoxyadenosine 5'-triphosphate were substituted for ATP in the Ca2+ pumping cycle of the sarcoplasmic reticulum Ca(2+)-ATPase. The rate of phosphorylation of the enzyme decreased by more than an order of magnitude when either of the hydroxyl groups was eliminated from the ribose ring. This resulted in low rates of hydrolysis and low levels of phosphoenzyme intermediate. In addition, the Km(1) of hydrolysis and the K1/2 of phosphorylation of the derivatives modified in the 3' position were decreased by a factor of 5-10. Otherwise, the 3'-amino-3'-deoxyadenosine 5'-triphosphate was utilized in a manner equivalent to ATP. Because the observed rates of phosphoenzyme formation with the deoxynucleotides were lowered to the extent that they would be rate-limiting in the enzyme cycle, and the level of phosphoenzyme intermediate remained low when the enzyme was back-inhibited by high Ca2+ concentrations, it was concluded that the majority of the enzyme remained in a preliminary conformation, in which the phosphorylation reaction could not proceed although substrate and Ca2+ were bound. It was then proposed that, following Ca(2+)-induced changes in conformation, the hydroxyl groups are able to form hydrogen bonds with pertinent segments of the phosphorylation domain, helping to stabilize an enzyme-substrate complex, one function of which may be to provide the proper stereochemistry for phosphate transfer.
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PMID:Elimination of the hydroxyl groups in the ribose ring of ATP reduces its ability to phosphorylate the sarcoplasmic reticulum Ca(2+)-ATPase. 846 22

2'-Deoxyadenosine 3'-tetraphosphate (2'-deoxy-3'-A4P) and 2', 5'-dideoxyadenosine 3'-tetraphosphate (2',5'-dideoxy-3'-A4P) were synthesized, and their effects were tested on crude and purified forms of native adenylyl cyclases isolated from brain. Syntheses combined the method of alkoxide activation with the use of tribromoethyl phosphoromorpholino-chloridate as an initial phosphorylating agent. Inhibition of adenylyl cyclase was rapid in onset. With 2'-d-3'-A4P or 2',5'-dd-3'-A4P inhibition of a purified native enzyme conformed to a linear noncompetitive behavior with respect to substrate, metal-5'ATP. Order of potency was 2', 5'-dideoxy- > 2'-deoxyadenosine and 3'-tetraphosphate > 3'-triphosphate. Both mechanism of inhibition and rank order of potency were consistent with inhibition via the 3'-nucleotide-(P)-site on adenylyl cyclase. Neither 2',5'-dd-3'-ATP nor 2',5'-dd-3'-A4P had any effect on the activities of other adenosine nucleotide binding proteins such as Ca2+/calmodulin-sensitive cyclic nucleotide phosphodiesterase, Na+/K+-ATPase, or cAMP-dependent protein kinase. With purified adenylyl cyclase from bovine brain 2',5'-dd-3'-A4P and 2'-d-3'-A4P gave, respectively, IC50 values of 9.3 and 15 nM and Ki values of 23 and 53 nM. These 3'-nucleotides are the most potent regulators described for adenylyl cyclases.
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PMID:Adenine nucleoside 3'-tetraphosphates are novel and potent inhibitors of adenylyl cyclases. 973 5

Stimulation of receptors for either ATP or adenosine leads to physiologic changes in retinal pigment epithelial (RPE) cells that may influence their relationship with the adjacent photoreceptors. The ectoenzyme nucleoside-triphosphate diphosphohydrolase-1 (NTPDase1) catalyzes the dual dephosphorylation of ATP and ADP to AMP. Although NTPDase1 can consequently control the balance between ATP and adenosine, it is unclear how its expression and activity are regulated. Classic negative feedback theory predicts an increase in enzyme activity in response to enhanced exposure to substrate. This study asked whether exposure to ATP increases NTPDase1 activity in RPE cells. Although levels of NTPDase1 mRNA and protein in cultured human ARPE-19 cells were generally low under control conditions, exposure to slowly hydrolyzable ATPgammaS led to a time-dependent increase in NTPDase1 mRNA that was accompanied by a rise in levels of the functional 78-kDa protein. Neither NTPDase2 nor NTPDase3 mRNA message was elevated by ATPgammaS. The ATPase activity of cells increased in parallel, indicating the up-regulation of NTPDase1 was functionally relevant. The up-regulation of NTPDase1 protein was partially blocked by P2Y1 receptor inhibitors MRS2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate] and increased by P2Y1 receptor agonist MRS2365 [(N)-methanocarba-2MeSADP]. In conclusion, prolonged exposure to extracellular ATPgammaS increased NTPDase1 message and protein levels and increased ecto-ATPase activity. This up-regulation reflects a feedback circuit, mediated at least in part by the P2Y1 receptor, to regulate levels of extracellular purines in subretinal space. NTPDase1 levels may thus serve as an index for increased extracellular ATP levels under certain pathologic conditions, although other mechanisms could also contribute.
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PMID:Stimulation of the P2Y1 receptor up-regulates nucleoside-triphosphate diphosphohydrolase-1 in human retinal pigment epithelial cells. 1762 96