UNIPROT:P50583 - FACTA Search

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Query: UNIPROT:P50583 (asymmetrical)
12,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) has been purified approx. 1500-fold from calf thymus. This enzyme phosphorylates 5'-hydroxyl termini in DNA using ATP as phosphate donor. RNA is phosphorylated at a much lower rate than DNA. The reaction requires the presence of a divalent cation, preferably Mg2+ or Mn2+ and is sensitive to sulfhydryl antagonists. The optimum pH for enzyme activity is 5.5. Enzyme activity is inhibited by low concentrations of inorganic sulfate and by some sulfate polymers. The kinase-catalyzed incorporation of the terminal phosphate of ATP into polynucleotides is inhibited by other nucleoside and deoxynucleoside triphosphates. The enzyme molecule has a molecular weight of about 70 000 and a Stokes radius of 4.3 nm. It has a frictional ratio of 1.44 indicating an asymmetrical structure. Calf thymus tissue should provide a useful alternative source for preparation of mammalian polynucleotide kinase.
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PMID:Purification and properties of polynucleotide kinase of calf thymus. 2 43

Erythroycte ghosts fixed in glutaraldehyde were dehydrated in (a) alcohol or acetone, (b) propylene glycol followed by Epon and embedded in an epoxy resin. A water-soluble urea/glutaraldehyde mixture was also used. The aim was to study the structure of the peripheral protein layer, which contains spectrin and actin, in the absence of OsO4 induced denaturation changes. Ghost membranes prepared in this way had an asymmetrical quadrilaminar structure. A layer of amorphous peripheral protein +/- 18 nm in width covered the entire inner face of the membrane in the form of a coarse meshwork in both Wash I (haemoglobin-containing) and haemoglobin-free ghosts. Cations (Mg2+ or Ca2+, or Mg2+ plus ATP) had no apparent effect on its fine structure. In contrast, the corresponding layer in OsO4-fixed membranes was represented by scanty, fuzzy material attached to the unit membrane only at irregular intervals. The results demonstrate the superior ability of glutaraldehyde to preserve the peripheral protein layer in thin sections, and afford further support for the view that much of this protein normally exists in an unpolymerized state.
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PMID:The ultrastructural organization of the contractile peripheral protein layer of the human erythroycte membrane. 10 28

1. Two mutants of the sodium channel II have been expressed in Xenopus oocytes and have been investigated using the patch-clamp technique. In mutant E387Q the glutamic acid at position 387 has been replaced by glutamine, and in mutant D384N the aspartic acid at position 384 has been replaced by asparagine. 2. Mutant E387Q, previously shown to be resistant to block by tetrodotoxin (Noda et al. 1989), has a single-channel conductance of 4 pS, that can be easily measured only using noise analysis. At variance with the wild-type, the open-channel current-voltage relationship of mutant E387Q is linear over a wide voltage range even under asymmetrical ionic conditions. 3. Mutant D384N has a very low permeability for any of the following ions: Cl-, Na+, K+, Li+, Rb+, Ca2+, Mg2+, NH+4, TMA+, TEA+. However, asymmetric charge movements similar to the gating currents of the Na(+)-selective wild-type are still observed. 4. These results suggest that residues E387 and D384 interact directly with the pathway of the ions permeating the open channel.
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PMID:Single point mutations of the sodium channel drastically reduce the pore permeability without preventing its gating. 166 Mar 94

1. The effects of Mg2+ on the single-channel conductance of neuronal nicotinic acetylcholine receptors were examined using receptors expressed by the rat phaeochromocytoma cell line, PC12. PC12 cells express at least three conductance classes of channels that are activated by acetylcholine, the largest conductance class being the most prevalent. This receptor channel is blocked by intracellular and extracellular Mg2+. 2. The effects of Mg2+ are asymmetrical; at a given concentration, internal Mg2+ is more effective at blocking outward currents than external Mg2+ is at blocking inward currents. Receptor channels are blocked at concentrations of Mg2+ that are low compared to the concentration of the main permeant cation, Na+, and the block is voltage dependent. 3. The block by Mg2+ is not complete as Mg2+ can permeate the channel. With 80 mM-extracellular Mg2+ (no extracellular Na+), the channel has an inward slope conductance of 2.9 pS. 4. The block by extracellular Mg2+ can be described by a one site, two barrier model for the channel which includes a negative surface charge on the external surface of the membrane. The parameters of the model place the binding site for Mg2+ at 52% of the membrane field from the outside with an apparent dissociation constant of 14 mM. However, the same parameters cannot describe the block by intracellular Mg2+. The deviations from the model suggest that the receptor channel may have more than one binding site for Mg2+.
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PMID:Voltage-dependent block by magnesium of neuronal nicotinic acetylcholine receptor channels in rat phaeochromocytoma cells. 172 94

The cuticle of the gill lamina of the crayfish Astacus leptodactylus (E), mechanically isolated, was mounted in an Ussing chamber and examined for its electrical properties. The cuticle of the gill lamina obtained from exuviae had similar properties. When perfused with artificial fresh water (AFW) outside and Van Harreveld solution (VH) inside, the transcuticular potential Voi was negative with respect to the inside, and close to the equilibrium potential for Cl- (ECl-). CH3COO-, HCO3-, SO4(2-) and cations (Na+, K+, Ca2+, Mg2+ and NH4+) behaved as impermeant ions with respect to Cl-. A decrease of pH (brought about with CO2) from 8.5 to 6.0 in AFW, VH or both had no effect on the potential. The cuticle area specific conductance was 20-30 mS/cm2 when superfused with AFW outside and VH inside. The conductance decreased linearly with log [Cl-] when Cl- was replaced by CH3COO-. Rectification was obvious when internal Cl- was reduced to 5 mmol/l. The Cl- selectivity of the cuticle could also be demonstrated in perfusing the cuticle with a single salt (NaCl, KCl, CaCl2, MgCl2 or LaCl3) and in diluting that salt on one side of the preparation or in replacing Cl- by CH3COO-, SO4(2-) and HCO3-. The potential changed almost linearly with log [Cl-] and was close to ECl-. The inner face of the cuticle was found to be slightly less selective than the outer face. The relative permeabilities were calculated to be: PCl- = 1, PNa+ = 0.001, PHCO3- = 0.0006, PCH3COO- = 0.0002. The dilution of a Cl- -free salt resulted in a cationic potential. The relative permeabilities of cations (NH4+, K+, Na+, Ca2+ and Mg2+) were found to range within a factor 2. The permeability of the cuticle to HCO3-, CH3COO- and SO4(2-) was 2-5 times lower. The cuticle conductance was linearly related to the activity of the salt perfusing the two sides of the preparation at equal concentrations. The molar area specific conductance to chloride salts was 14 (mS/cm2)/(mmol/l). That of Cl- -free salts ranged from 1 to 20 (microS/cm2)/(mmol/l) depending on the salt used. It was deduced that PCl- is 2 X 10(-3) cm/s and that all the other ions tested have permeabilities of 10(-7)-10(-6) cm/s. With large intensity current pulses the cuticle exhibited rectifying properties and an asymmetrical behaviour. Increasing the pH of the perfusing solution reduced the transcuticular potential established with a Cl- gradient.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Ionic permeabilities of the gill lamina cuticle of the crayfish, Astacus leptodactylus (E). 241 Jun 7

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.
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PMID:Identification and partial characterization of an adenosine(5')tetraphospho(5')adenosine hydrolase on intact bovine aortic endothelial cells. 254 89

The P1P4-bis(5'-nucleosidyl) tetraphosphate asymmetrical-pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia has been purified over 11,000-fold to homogeneity. Anion-exchange chromatography resolves two major species with very similar properties. The enzyme is a single polypeptide of Mr 17,600 and is maximally active at pH 8.4 and 2 mM-Mg2+. It is inhibited by Ca2+ (IC50 = 0.9 mM with 2 mM-Mg2+) but not by Zn2+ ions. It preferentially hydrolyses P1P4-bis(5'-nucleosidyl) tetraphosphates, e.g. P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) (kcat. = 12.7 s-1; Km = 33 microM) and P1P4-bis(5'-guanosyl) tetraphosphate (Gp4G) (kcat. = 6.2 s-1; Km = 5 microM). With adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) as substrate, there is a 4.5-fold preference for AMP and GTP as products and biphasic reaction kinetics are observed giving Km values of 4.7 microM and 34 microM, and corresponding rate constants of 6.5 s-1 and 11.9 s-1. The net rate constant for Ap4G hydrolysis is 7.6 s-1. The enzyme will also hydrolyse nucleotides with more than four phosphate groups, e.g. Ap5G, Ap6A and Gp5G are hydrolysed at 25%, 18% and 10% of the rate of Ap4A respectively. An NTP is always one of the products. Ap2A and Gp2G are not hydrolysed, while Ap3A and Gp3G are very poor substrates. When the enzyme is partially purified from embryos and larvae at different stages of development by sedimentation through a sucrose density gradient, its activity increases 3-fold during the first 12 h of pre-emergence development. This is followed by a slow decline during subsequent larval development. The similarity of this enzyme to other asymmetrical-pyrophosphohydrolases suggests that it did not evolve specifically to degrade the large yolk platelet store of Gp4G which is found in Artemia embryos, but that it probably serves the same general function in bis(5'-nucleosidyl) oligophosphate metabolism as in other cells.
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PMID:Characterization of the bis(5'-nucleosidyl) tetraphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia. 254 71

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

Homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase (Ap4A-phosphorylase), the enzyme recently found in yeast (Guranowski, A., and Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547) catalyzes an exchange reaction between the beta-phosphate of nucleoside diphosphate (NDP) and orthophosphate from the medium (Pi). The common purine and pyrimidine ribonucleoside diphosphates as well as ADP analogs modified either in aglycone, sugar, or at the anhydride bond beta-position are substrates. The Km and rate values for the NDP-Pi exchange reaction were estimated at pH optima. These optima are 6.5 for UDP, 7.0 for ADP or CDP, and 8.0 for GDP. In the presence of 10 mM K2HPO4, 0.1 mM EDTA, and 100 mM Hepes/KOH (pH 7.0), the Km for ADP is 0.7 mM with a rate constant at saturating ADP of 96 s-1. The Km value for orthophosphate is 2 mM. In the NDP-Pi exchange reaction, phosphate can be substituted with arsenate and apparent arsenolysis of NDPs yields corresponding nucleoside monophosphates. The same pH optimum of 6.5 is found for arsenolysis of ADP, GDP, and CDP. Whereas the Ap4A phosphorylase sulfhydryl groups are essential for catalyzing the Ap4A phosphorolysis, the NDP-Pi exchange reactions, and the arsenolysis of NDPs, the divalent metal ions (Mg2+, Mn2+, Ca2+, Co2+, and Cd2+), which had been shown to be essential cofactors of the former reaction, are not required for the two latter ones. Used at concentrations which are optimum for Ap4A phosphorolysis, the cations (particularly Mg2+ and Cd2+) inhibit the NDP-Pi exchange and the arsenolysis of NDPs. Interestingly, the Ap4A phosphorylase exhibits higher specificity for adenosine 5'-phosphosulfate (APS) than for any other NDP tested. The V/Km ratio is almost 5-fold higher with APS than with ADP. However, in the presence of orthophosphate, the APS is irreversibly converted to ADP. Thus, the enzyme displays a property already attributed to ADP-sulfurylase (EC 2.7.7.5), (Grunberg-Manago, M., Del Campillo-Campbell, A., Dondon, L., and Michelson, A. M. (1966) Biochim. Biophys. Acta 123, 1-16; Nicholls, R. G. (1977) Biochem. J. 165, 149-155).
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PMID:Diadenosine 5',5'''-P1, P4-tetraphosphate alpha, beta-phosphorylase from yeast supports nucleoside diphosphate-phosphate exchange. 300 35

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