Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P50583 (asymmetrical)
 12,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.
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PMID:Yeast diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase. 282 98

Novel enzymatic activity which splits diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorolytically has been found in extracts from Saccharomyces cerevisiae. One of the two alpha,beta-anhydride bonds between Ap4A phosphate residues undergoes phosphorolysis, and ATP (pppA) plus ADP (ppA) are the products of the reaction according to the equation: AppppA + P*i----pppA + p*pA The reaction is dependent on the presence of divalent metal ions; Mn2+ or Mg2+ sustain the greatest rates of reaction. Among analogues of the Ap4A substrate, Ap5A and Gp4G, but not p4A and Ap3A, are substrates, and corresponding products are p4A plus ADP, and GTP plus GDP, the phosphate being incorporated into the nucleoside 5'-diphosphates. In the reactions, phosphate can be substituted with arsenate. Arsenolysis of Ap4A, Ap5A, or Gp4G leads to ATP plus AMP, p4A plus AMP, and GTP plus GMP, respectively. The name diadenosine tetraphosphate alpha,beta-phosphorylase (ADP-forming) is proposed for the new enzyme. The phosphorylase has been purified to apparent homogeneity and behaves as a single polypeptide chain of Mr = 40,000. Optimum activity of the enzyme is at pH 8.0 and the sulfhydryl groups are essential for catalysis. At saturating Ap4A, the rate constant for the reaction is 36 s-1 and the Km value for Ap4A is 60 microM (37 degrees C, 50 mM Hepes/KOH (pH 8.2), 500 microM MnCl2, 10 mM K2HPO4, 1 mM 2-mercaptoethanol, and 2% glycerol). The Km values for phosphate and arsenate are 1 and 3 mM, respectively.
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PMID:Phosphorolytic cleavage of diadenosine 5',5'''-P1,P4-tetraphosphate. Properties of homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase from Saccharomyces cerevisiae. 298 63

We found that the lengths of all sarcomeres spontaneously oscillated in an isolated skeletal myofibril, when both ends were fixed, submillimolar to millimolar concentrations of ATP, ADP and inorganic phosphate (Pi) were present, and Ca2+ was removed. Narrowing and widening of an H-zone and an I-band were observed corresponding to the shortening and lengthening of a sarcomere, suggesting that thick and thin filaments slide past each other. The oscillation of each sarcomere was asymmetrical, consisting of a rapid lengthening phase and a slow shortening phase. The period of oscillation was about 3s; the peak-to-peak amplitude of oscillation reached as much as 30% of the average sarcomere length. The propagation of the sarcomere oscillation along the long axis of the myofibril was observed occasionally in single myofibrils and frequently in bundles of myofibrils. The 'state'-diagram showing the concentration range of ADP and Pi in which contraction, oscillation or relaxation of myofibrils occurs in the presence of ATP and the absence of Ca2+ suggested that the oscillation is a third state of skeletal muscle located in between the contracting and relaxing states.
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PMID:Spontaneous oscillatory contraction of sarcomeres in skeletal myofibrils. 313 84

The synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.
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PMID:A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver. 364 96

The properties of SH-groups of mitochondrial creatine kinase existing in solution as a hexamer with Mr of (240 +/- 12) X 10(3) Da, were investigated. The number and reactivity of SH-groups by specific modifiers--[5.5'-dithiobis-(2-nitrobenzoic acid), DTNB; 7-chloro-4-nitrobenzo-2-oxo-1.3-diazol, NBD-Cl; 2.2'-dithiopyridine, DTP] were determined. It was found that each subunit of the enzyme hexameric molecule contains two modified SH-groups, only one of which is protected against modification by Mg-ADP, Mg-ATP as well as during the formation of the transition state analog (TSA)--E-Mg X ADP-NO3-creatine--and is essential for the enzyme activity. These six essential SH-groups within the hexameric molecule of mitochondrial creatine kinase may be classified into two groups according to the rate of their interaction with DTNB, NBD-Cl and DTP. The rate constants of modification of three fast and three slow essential SH-groups differ 4-10 times. The kinetics of enzyme inactivation by iodoacetamide (IAA) is biphasic; each phase is characterized by a 50% loss of activity. The inactivation constants differ 30 times; both phases being protected by TSA; consequently, the inactivation is caused by the binding of IAA to the essential SH-groups. The unequal reactivity of essential SH-groups seems to be preexisting. Using a computer analysis, the dependence of the amount of residual activity on the number of modified SH-groups by NBD-Cl and DTNB was studied. The interaction of NBD-Cl and DTNB with the most reactive essential SH-groups in half of the subunits results in the inactivation of these subunits as well as in partial or complete inactivation of the other half of the non-modified subunits. The degree of inactivation of the latter 50% of subunits strongly depends on the nature of the modifier. The inactivating effect of the bound modifier is translated from one subunit to another in one direction. The experimental results point to asymmetrical association of mitochondrial creatine kinase subunits.
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PMID:[Non-equivalency of SH groups essential for the activity of mitochondrial creatine kinase]. 369 21

In order to elucidate the postulated role of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) in cell growth regulation, the Ap4A cellular content was measured in cells submitted to various treatments affecting the cell growth. Ap4A level was found to increase ten times when cells reached confluence, whereas no significant variation of the ATP pool was observed. Cell growth arrest after serum depletion did not cause any variation in the Ap4A pool. A limited increase in the Ap4A pool was observed when growth of arrested cells was reinitiated but this variation reflected only the increase of cell density. No significant variation in the Ap4A intracellular level was observed after submitting two eukaryotic cell lines to various stresses (cytotoxic drugs, ethanol and heat-shock treatments). These results suggest that, in eukaryotic cells, Ap4A is not involved in cell growth stimulation but rather is associated with cell contact growth inhibition. They also suggest that Ap4A is not an 'alarmone', contrary to what has been proposed for bacteria.
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PMID:Relationship between cellular diadenosine 5',5'''-P1,P4-tetraphosphate level, cell density, cell growth stimulation and toxic stresses. 375 97

Ap4A levels in sperms, eggs and different developmental stages of sea urchin (Psammechinus miliaris) and (Xenopus laevis) were determined by a method based on ATP measurement with luciferin/luciferase after splitting diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) into ATP and AMP. Appreciable storage pools of Ap4A were found in unfertilized eggs of Psammechinus and Xenopus as well as in sea urchin sperms. The actual Ap4A concentration of 28 microM in sperm represents the highest Ap4A level so far observed in eukaryotic cells. Upon fertilization an instant onset of de novo synthesis of Ap4A was demonstrated. Ap4A levels during early embryogenesis of P. miliaris and X. laevis (2.5-4 microM) are higher than those in exponentially growing mammalian culture cells and mammalian fetuses. Microinjection of Ap4A into unfertilized eggs of Psammechinus miliaris caused a 3-7 fold increase of DNA synthesis in comparison with mock-injected eggs.
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PMID:High diadenosine tetraphosphate (Ap4A) level in germ cells and embryos of sea urchin and Xenopus and its effect on DNA synthesis. 404 45

1. ADP/ATP transport has been reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [14C]ADP uptake was strictly dependent on internal ATP. 2. The simplest phospholipid system capable of yielding high rates of ADP/ATP transport was a mixture of phosphatidylethanolamine and cariolipin (92: 8, w/w). 3. ADP/ATP transport in the reconstituted system proceeded by exchange-diffusion with a 1/1 stoichiometry. The specificity for aDP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20 degrees C, at 20 mM internal ATP, routinely ranged between 300 and 1000 nmol of nucleotide exchanged per min/mg of added carrier protein. The apparent Km value for external ADP was around 10 microM. 4. The ADP/ATP exchange in the reconstituted system was rather stable to ageing. It dropped by only 20% after 1 day of ageing at 20 degrees C. Divalent cations (Mg2+, Mn2+, Ca2+) at concentrations higher than 1 to 2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. 5. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in the mitochondria. 6. The percentage of competent carrier protein in liposomes was calculated from dose-response data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [3H]-atractyloside removable by ADP. By both methods, 3 to 6% of the added carrier protein was found to be competent in ADP/ATP transport, based on the assumption that the binding of one atractyloside or carboxyatractyloside molecule per 30000 molecular weight carrier unit results in complete inhibition of transport. 7. Freeze-fracture electron microscopy showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesicles (2 to 4% depending on the amount of carrier protein added) were clearly particulated. About 90% of the particulated vesicles showed no more than 2 particles per vesicle and only 5% more than 5 particles per vesicle. The distribution of the particles between convex and concave fracture faces was asymmetric; about 2/3 of the protein molecules were anchored at the external surface of the vesicles and only 1/3 at the internal one...
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PMID:Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles. 625 72

Native and modified phenylalanine transfer ribonucleic acid (tRNAPhe) can modulate phenylalanine-dependent adenosine triphosphate--inorganic [32P]pyrophosphate (ATP--[32P]PPi) exchange activity via inhibition of adenylate synthesis. Inhibition is visualized if concentrations of L-phenylalanine, ATP, and pyrophosphate are subsaturating. In the proposed mechanism, tRNAPhe is a noncompetitive inhibitor at conditions where only one of the two active sites per molecule of enzyme is occupied by L-phenylalanine, ATP, and pyrophosphate. At saturating concentrations of these reactants, both active sites are occupied and, according to the model, inhibition is eliminated. Occupation by these reactants is assumed to follow homotropic negative cooperativity. The type of effects depends on modification of tRNAPhe. Native tRNAPhe, tRNA2'-dAPhe, and tRNAoxi-redPhe are inhibitors, tRNAPhepCpC has no effect, and tRNAoxPhe is an activator. Kinetics of activation by tRNAoxPhe are slow, following the time course of Schiff base formation and subsequent reduction by added cyanoborohydride. Besides showing that a putative enzyme amino group is nonessential for substrate binding and adenylate synthesis, this result may suggest that an enzyme amino group could interact with the 3'-terminal adenyl group of cognate tRNA. In the case of asymmetrical occupation of the enzyme active sites by all of the small reactants ATP, L-phenylalanine, and pyrophosphate, the interaction with the amino group might trigger the observed noncompetitive inhibition of the pyrophosphate exchange by tRNAPhe.
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PMID:Phenylalanyl-tRNA synthetase of baker's yeast. Modulation of adenosine triphosphate-pyrophosphate exchange by transfer ribonucleic acid. 626 48

An enzyme hydrolyzing diadenosine 5',5"'P1, P4-tetraphosphate (Ap4A) to AMP and ATP has been purified to apparent homogeneity from mouse liver cell extracts. The isolation procedure comprised ammonium sulfate precipitation, chromatography on Sephadex G-75. DEAE-cellulose, blue Sepharose and AMP-Sepharose. The enzyme is a single polypeptide chain with a native Mr = 64,000 with a Km of 1.66 microM and Vmax of 1.25 mumol/min. AMP, ADP, Ap4, GTP, Gp4, Ap3A, Ap5A, Gp3G, and Gp5G are noncompetitive inhibitors of the Ap4A hydrolase activity, whereas Gp4G inhibits Ap4A hydrolysis competitively with a Ki of 6 microM. Theophylline, caffeine, and isobutylmethylxanthine do not or only slightly inhibit Ap4A hydrolysis. Mitogenic factors have no effect on the enzymatic activity of Ap4A hydrolase, excluding that a direct influence of internalized mitogens on Ap4A degradation could be responsible for mitogen-dependent fluctuation of intracellular Ap4A pool sizes.
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PMID:Diadenosine tetraphosphate hydrolase from mouse liver. Purification to homogeneity and partial characterization. 627 21


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