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)

The effects of amino acid substitutions in the carboxyl terminal region of the H(+)-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263----end, Trp-231----end, Glu-219----Gln, and Arg-210----Lys (or Gln) were fully defective in ATP-dependent proton translocation, and those with Gln-252----Glu (or Leu), His-245----Glu, Pro-230----Leu, and Glu-219----His were partially defective. On the other hand, the phenotypes of the Glu-269----end, Ser-265----Ala (or end), and Tyr-263----Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation.
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PMID:Role of the carboxyl terminal region of H(+)-ATPase (F0F1) a subunit from Escherichia coli. 182 13

The Schizosaccharomyces pombe nuclear gene, atp2, encoding the beta subunit of the mitochondrial ATP synthase, was sequenced and found to contain a 1575-bp open reading frame. Two adjacent transcription-initiation sites were found at positions 34 and 44 nucleotides upstream of the translation-initiation codon. The deduced polypeptide sequence was composed of 525 amino acid residues (molecular mass = 56875 Da). The mature polypeptide starts at residue 45 (molecular mass = 51,685 Da), indicating the presence of a presequence of 44 residues, presumably involved in mitochondrial targeting. The atp2 mutant B59-1 [Boutry, M. & Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477] and its related revertant allele R4-3 [Jault, J. M., Di Pietro, A., Falson, P., Gautheron, D. C., Boutry, M. & Goffeau, A. (1989) Biochem. Biophys. Res. Commun. 158, 392-399] were also cloned and sequenced. A single nonsense mutation, CAG (Gln170)----TAG (stop) in mutant B59-1, became a missense mutation, TAG (stop)----TAC (Tyr) in revertant R4-3. Gln170 is located between the first and second elements belonging to the nucleotide-binding site. Its substitution by a tyrosine residue increases the enzyme affinity towards ADP, the amount of endogenous nucleotides and the apparent negative cooperativity for ATPase activity.
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PMID:Beta subunit of mitochondrial F1-ATPase from the fission yeast. Deduced sequence of the wild type protein and identification of a mutation that increases nucleotide binding. 183 60

Depletion of intracellular calcium stores appears to increase plasma membrane permeability for calcium by an as yet obscure mechanism. We found that the Ca2+ ionophore, A23187, and thrombin elevate cytosolic calcium ([Ca2+]i) equally and cause tyrosine phosphorylation of a 130-kDa protein and to a lesser extent 80- and 60-kDa proteins. Chelation of [Ca2+]i by 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid/acetomethoxy ester decreased thrombin-induced tyrosine phosphorylation responses. These results suggested that [Ca2+]i elevation promotes tyrosine phosphorylation. Tyrosine phosphorylation persisted in the presence or absence of extracellular calcium after thrombin stimulation but subsided rapidly after A23187 addition if extracellular calcium was present. When Ca2+/ATPase activity, which is apparently required to maintain calcium stores, is inhibited by low temperature, tyrosine phosphorylation of the 130-kDa protein occurs. Rewarming platelets reverses tyrosine phosphorylation only if extracellular calcium is present. Thapsigargin, a calcium ATPase inhibitor, also induces tyrosine phosphorylation of the 130-kDa protein and prevents dephosphorylation of this protein when added prior to rewarming. These observations suggest that homeostatic levels of calcium in storage compartments favor tyrosine dephosphorylation of specific proteins. Thus the levels of [Ca2+]i and stored calcium appear to control tyrosine phosphorylation antagonistically. Tyrosine phosphorylation may play a role in regulating calcium channel function.
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PMID:Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins. 183 59

1. Calcium-stimulated ATPase-ADPase activities were studied in a microsomal fraction of rat placental tissue. 2. The kinetic characteristics correspond to those of ATP-diphosphohydrolase, also known as apyrase (E.C. 3.6.1.5). 3. These characteristics include the lack of specificity towards nucleoside di- and triphosphates, activation by Ca2+, Mg2+ or Mn2+, insensitivity to specific inhibitors of some ATPase and absence of an effect of sulphydryl reagents. 4. Chemical modification of tyrosine, tryptophan, arginine and carboxylic residues decreases both ATPase and ADPase activities. 5. The substrate analogue, 5'-(beta, gamma-methylene)triphosphate, protected both enzyme activities against all the modifying amino acid reagents tested. 6. Placental fractions (homogenate and microsomes) inhibit ADP-dependent platelet aggregation. 7. The solubilized microsomal enzyme has a molecular mass of 67 kDa by size-exclusion chromatography; the pI is 9.36. 8. A differential effect is observed on the activation produced by Concanavalin A on microsomal and solubilized fractions when treated in the presence and absence of alpha-methylmannoside.
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PMID:ATPase-ADPase activities of rat placental tissue. 183 61

A synthetic, 28-residue peptide derived from the calmodulin-binding sequence of the plasma membrane Ca2+ pump (C28W) inhibits the ATPase activity of a calpain-produced, truncated fragment of the enzyme. The fragment, which has lost the calmodulin-binding domain, has a molecular mass of 124 kDa and is fully active in the absence of calmodulin. Replacement of Trp-8 in the peptide by an Ala decreases the overall inhibitory activity, while replacement with a Tyr increases it. However, at very low peptide concentrations the effect of Tyr replacement disappears. The synthetic peptide has been made photoactivatable by replacing Phe in position 9 with a synthetic phenylalanine analogue containing a diazirine group and was radioactively labeled by coupling a [3H]acetyl function to its N terminus. After cross-linking with the derivatized peptide, the 124-kDa fragment has been proteolyzed with either Lys-C, Asp-N, or V8 proteases, and the fragment(s) have been separated. Partial sequencing of the cross-linked, radioactive peptides has identified a site of the pump located C terminally to the phosphoenzyme-forming aspartic acid, spanning residues 537-544 of the hPMCA4 isoform of the enzyme. It is concluded that this sequence is part of a site which binds the calmodulin-binding domain of the pump.
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PMID:The plasma membrane Ca2+ pump contains a site that interacts with its calmodulin-binding domain. 184 39

Long-term amiodarone therapy is invariably associated with some side effects. Although its mechanism of action, as an antiarrhythmic drug is well understood, the side effect profile of amiodarone is not yet established. To determine possible mechanisms, the interaction of amiodarone and its major metabolite desethylamiodarone with calmodulin was investigated, since calmodulin is known to regulate Ca2+ transport, cell proliferation and the enzymes involved in signal transduction and nucleotide metabolism. The interaction between the drugs and calmodulin was studied by monitoring intrinsic tyrosine fluorescence of calmodulin and by using a fluorescent probe, N-phenyl-1-naphthylamine (NPN). 14C-Chlorpromazine displacement studies were conducted to differentiate the specific binding sites. The effect on the biological activity of calmodulin was determined with calmodulin dependent phosphodiesterase and Ca2(+)-ATPase. The dansyl calmodulin was used as fluorescent probe to study the effect of these drugs on complex formation between calmodulin and phosphodiesterase. Both amiodarone and desethylamiodarone decreased tyrosine fluorescence of calmodulin with IC50 of 4.9 and 4.4 microM respectively and these interactions were Ca2(+)-dependent. NPN fluorescence was also affected in a concentration dependent manner. These drugs also displaced bound 14C-chlorpromazine from calmodulin and the effect was biphasic. However, desethylamiodarone was more potent than amiodarone. The binding of 3H-amiodarone to calmodulin was modified by a variety of compounds, one class of compounds decreased and the other increased 3H-amiodarone binding to calmodulin. Only, desethylamiodarone inhibited the phosphodiesterase activation by calmodulin with IC50 of 13.2 microM without changing the basal enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of calmodulin properties by amiodarone and its major metabolite desethylamiodarone. 184 30

Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C. 196 40

The main characteristics of L-tyrosine (L-Tyr) uptake by B16/F10 malignant melanocytes are reported. This amino acid can be taken up by two systems, both of them being saturable. The first one would be system L. This system can be studied in cells preloaded with amino acids that are a good substrate for system L, such as L-methionine or L-tryptophan. The kinetic parameters for L-Tyr uptake by this transport system are Vm = 6.5 pmol L-Tyr/10(3) cells.min and Km around 130 microM. The second system, probably the system ASC, shows lower capacity but higher affinity than the former. This system can be detected only in cells previously depleted of amino acids, showing approximate kinetic values of Vm 0.05 pmol L-Tyr/10(3) cells.min and Km around 5 microM. It is shown that the increase in cell density yields a decrease in the rate of L-Tyr uptake by system L, but this increase does not affect the high affinity system, alpha-MSH does not affect significantly the L-Tyr uptake by both systems. 2-Amino bicyclo-(2,2,1)-heptane-2-carboxylic acid produces a remarkable inhibition of the rate of L-Tyr uptake, but alpha-methylaminoisobutyric acid does not affect the rate of transport of this amino acid. The absence of sodium produces a slight but reliable decrease in the rate of L-Tyr uptake, supporting the involvement of two different transport systems. The ionophores monensin and nigericin enhance the transport by system L, but this effect is suppressed by the presence of ouabain. This finding indicates that the (Na+ -K+)-ATPase is essential for the stimulating action of ionophores.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transport of L-tyrosine by B16/F10 malignant melanocytes: characterization of the process. 198 30

The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 284, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl). The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D. and Hou, C. (1989) Biochim. Biophys. Acta 974, 24-29). We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site. NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent. Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits. The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1. The strict asymmetry of labeling of the free F1-ATPase was not observed. Thus, double labeling of beta subunits by several reagents was found. This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.
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PMID:Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits. 213 13

Using site-directed mutagenesis, Tyr-307, Tyr-341, or Tyr-364, supposedly located at the adenine nucleotide binding site(s) of the beta subunits of F1-ATPase from the thermophilic bacterium PS3, was replaced with Phe or Cys. The alpha 3 beta 3 complexes reconstituted from the alpha subunits and individual mutant beta subunits hydrolyzed ATP. Thus, neither the hydroxyl groups nor the aromatic rings in these positions are required for ATPase activity of F1-ATPase.
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PMID:Aromatic rings of tyrosine residues at adenine nucleotide binding sites of the beta subunits of F1-ATPase are not necessary for ATPase activity. 213 33


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