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

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

Reaction of rat liver metallothionein-II with two bifunctional cross-linking reagents, glutaraldehyde and dimethyl suberimidate, produces high yields of polymeric species. It is argued that cross-linking is trapping preformed aggregates of the protein, which therefore represent a stabilized quaternary structure of metallothionein. The two polymeric species differ in a number of respects. With dimethyl suberimidate, the polymer retains all metal-binding sites of the monomer, and has an unaltered isoelectric point. Reaction with glutaraldehyde causes loss of one or two Cd2+/Zn2+-binding sites and elevates the pI. Both species are nearly spherical aggregates, in contrast with the highly asymmetrical metallothionein. Both polymers are linked through lysine residues, and the thiol groups remain reduced. The biological significance of these aggregates is discussed.
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PMID:Chemical modifications of metallothionein. Preparation and characterization of polymers. 643 86

The effect of severe zinc deficiency on the distribution of nine elements (potassium, phosphorus, sodium, magnesium, calcium, iron, zinc, copper and manganese) in brain regions (olfactory lobes, right and left hippocampi, cerebellum and the rest of the brain) has been studied. After male rats (30 days old) were fed a zinc-deficient diet for 28 days, the zinc concentration of most brain parts was similar to zinc-adequate control values. Olfactory lobe zinc, on the other hand, was slightly depressed. However, the levels of other metals were dependent on zinc nutriture. Zinc deficiency caused an elevation in copper concentrations in most brain parts. Restriction of food intake caused a similar increase in brain copper but generally the effect was less than with zinc deficiency. Levels of calcium, manganese, sodium and potassium, in certain brain regions, also appeared to be altered by the zinc status of an animal. Of the minerals examined, only zinc and copper displayed asymmetrical distribution between the right and left hippocampus, and severe zinc deficiency did not affect lateral distribution of these trace metals in the hippocampus. The data suggest the hypothesis that changes in brain metal content, associated with zinc deficiency, contribute to the behavioral abnormalities that occur.
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PMID:Severe zinc deficiency: effects on the distribution of nine elements (potassium, phosphorus, sodium, magnesium, calcium, iron, zinc, copper and manganese) in regions of the rat brain. 661 70

Zinc, adenosine 5'-phosphate (AMP), and pyrophosphatase greatly stimulate the synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) by rat liver lysyl-tRNA synthetase. The synthesis of Ap4A does not require lysine; thus the lysyl-adenylate complex is not required. The substrates have been determined to be adenosine 5'-triphosphate (ATP) and AMP with apparent Km values of 2.1 mM and 1.5 mM, respectively. A zinc-dependent hydrolysis of ATP and AMP has been shown to be associated with the synthetase. In the presence of zinc there is a direct correlation between both the amount of AMP formed and the amount of Ap4A synthesized by lysyl-tRNA synthetase. Ap4A acts as a competitive inhibitor for ATP in the aminoacylation reaction of lysyl-tRNA synthetase with a KI of 2.5 microM. Concentrations of Ap4A up to 12.5 microM do not inhibit the synthesis of Ap4A by lysyl-tRNA synthetase. This suggests that there may be more than one binding site for ATP on the enzyme.
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PMID:Characterization of a homogeneous complex of arginyl- and lysyl-tRNA synthetase: zinc and adenosine 5'-phosphate dependent synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate. 662 5

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.
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PMID:Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus. 669 19

This article presents a fluorimetric study of the main properties of the enzymes dinucleoside tetraphosphate (asymmetrical) hydrolase or dinucleoside tetraphosphatase (Ap4Aase, EC 3.6.1.17) and dinucleoside triphosphate hydrolase or dinucleoside triphosphatase (Ap3Aase, EC 3.6.1.29), both present in adrenal medulla cytosolic extracts. Diethenoadenosine polyphosphates, epsilon-(ApnA), are used as artificial fluorogenic substrates. Ap4Aase exhibits a molecular mass around 20 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ and preferentially hydrolyzes substrates with four phosphate groups. Km for epsilon-(Ap4A) is 1.3 microM and Ki for Ap4A and Gp4G are 1 and 0.2 microM respectively. Km for Ap4A determined by HPLC is 1.6 microM. epsilon-(Ap5A) and epsilon-(Ap6A) are hydrolyzed at reduced rates. This enzyme is inhibited by Zn2+, F- and very strongly by Ap4 and epsilon-Ap4. Ca2+ cannot replace Mg2+, but behaves as inhibitor in its presence. The substrate analogs dinucleoside triphosphates Ap3A, G;3G, m7Gp3G and m7Gp3A and the periodate-oxidized nucleotides o-(Ap4A), o epsilon-(Ap4A), o-Ap4 and o epsilon-Ap4 behave as inhibitors. Ap3Aase exhibits a molecular mass around 30 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ or Ca2+, but retains a low measurable activity around 10% in the absence of these divalent cations. It only hydrolyzes substrates with three phosphate groups. Km for epsilon-(Ap3A) is 11 microM and Ki for Ap3A and Gp3G are 20 and 22 microM, respectively. Km for Ap3A determined by HPLC is 16 microM. m7Gp3G and m7Gp3A are also good substrates for triphosphatase.
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PMID:Specific dinucleoside polyphosphate cleaving enzymes from chromaffin cells: a fluorimetric study. 749 90

Carbapenem-hydrolyzing beta-lactamase from Serratia marcescens FHSM4055 was purified 926-fold by means of carboxylmethyl Sephadex C-50, Sephacryl S-200, and Mono S column chromatography. The molecular weight was 30,000 by SDS-PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p-chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo-beta-lactamase and that the SH-group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (kcat/Km) showed that the enzyme hydrolyzed various beta-lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino- and imino-groups, ceftazidime with a carboxypropyloxyimino-group, and cefclidin with a carbamoylquinuclidine-group were poor substrates among the beta-lactam antibiotics other than the monobactams tested. The plots of the turnover number (kcat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the 'tail' on the acid side (pK1, 5.9; pK2, 9.0; pK3, 10.8), whereas those of kcat/Km gave a bell-shaped curve (pK1, 5.8; pK2, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of metallo-beta-lactamase from Serratia marcescens. 778 75

Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase and Ap4A pyrophosphohydrolase activities have been purified from extracts of the green alga Scenedesmus obliquus. Both activities were also detected in Scenedesmus brasiliensis, Scenedesmus quadricauda and in Chlorella vulgaris. This is the first time that both types of enzyme have been detected in the same species. The Ap4A phosphorylase has a molecular mass of 46-48 kDa, a broad pH optimum between 7.5 and 9.5, and requires a divalent ion for activity (Mg2+ > Co2+ > Ca2+ = Mn2+ = Cd2+ > Zn2+). It degrades substrates with at least four phosphate groups and always produces a nucleoside 5'-diphosphate product. The Km values for Ap4A and Pi are 5.3 microM and 160 microM, respectively, and kcat. = 1.8 s-1. Arsenate, vanadate, molybdate, chromate and tungstate can substitute for phosphate. The enzyme also catalyses Ap4A synthesis (Keq. = [Ap4A] [Pi]/[ATP][ADP] = 9 x 10(-4)) and ADP arsenolysis. The Ap4A hydrolase has a molecular mass of 26-28 kDa, an alkaline pH optimum of 8.8-9.8, and prefers Zn2+ as the stimulatory ion (Zn2+ > Mg2+ > Mn2+ > Co2+ > Cd2+). It degrades substrates with at least four phosphate groups, having a slight preference for Ap5A, and always produces a nucleoside 5'-triphosphate product. The Km value for Ap4A is 6.6 microM and kcat. = 1.3 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 0.67 microM) and non-competitively by fluoride (Ki = 150 microM). A 50-54 kDa dinucleoside 5',5'''-P1,P3-triphosphate (Ap3A) pyrophosphohydrolase was also detected in S. obliquus, S. quadricauda and C. vulgaris. The corresponding enzyme in S. brasiliensis (> 100 kDa) may be a dimer
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PMID:The green alga Scenedesmus obliquus contains both diadenosine 5',5'''-P1,P4-tetraphosphate (asymmetrical) pyrophosphohydrolase and phosphorylase activities. 819 32

Transepithelial and cell membrane potential measurements have suggested that the basolateral membrane of gerbil vestibular dark cells contains Cl- conductive pathways. We used the patch clamp technique to search this membrane for Cl- conductive channels which could account for the macroscopic observations. Two types of Cl- channel were found in both cell-attached and excised membrane patches. One type was found with an incidence of 19% and had a single-channel conductance of 95 +/- 1 pS (N = 20) in symmetrical Cl- solutions. The other type was found with an incidence of 3% and had a large single-channel conductance of 360 +/- 11 pS (N = 12) in symmetrical Cl- solutions (LC-type Cl- channel). Both types of Cl- channel had linear current-voltage relations and at least 2 substates. In asymmetrical Cl- solutions (gluconate substitution) the current-voltage relations fit the Goldman-Hodgkin-Katz current equation for Cl-. Neither channel was blocked by Zn2+, NPPB, DIDS, DNDS or quinine. The 95 pS channel exhibited a spontaneous 'rundown' of its activity within 1 to 10 min after being excised. This rundown was not reversed by the catalytic subunit of protein kinase A. Channel activity was not dependent on the presence of cytosolic Ca2+ nor markedly altered by variations in cytosolic pH between 6.5 and 8.0. The two Cl- channels were distinguished by the membrane voltage ranges in which they were active and by their anion selectivity. The open probability of the 95 pS channel was insensitive to voltage and the anions NO3-, I- and Br- were only half as permeable as Cl-. By contrast, the LC-type Cl- channel was mostly active between about +/- 30 mV and equally permeable to NO3-, I-, Br- and Cl-. The 95 pS Cl- channel may account for the observed transepithelial and intracellular voltage responses to Cl- concentration steps and provide the path for the recirculation of Cl- across the basolateral membrane. The LC-type Cl- channel shows the same lack of anion discrimination as the anion pathway activated during hyposmotic challenge.
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PMID:Two types of chloride channel in the basolateral membrane of vestibular dark cells. 822 32

The diadenosine 5',5'''-P1,P4-tetraphosphate (asymmetrical) hydrolase (EC 3.6.1.17) from human placenta has been purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephacel, gel filtration on Sephadex G-100, and affinity elution from red Sepharose. The enzyme is a single polypeptide of M(r) 19,200. It exhibits maximum (100%) activity at pH 7.3 in the presence of 3 mM MgCl2 and 60, 50, and 40% of the activity in 1 mM CoCl2, 0.1 mM ZnCl2, and 0.5 mM MnCl2, respectively. The Km value calculated for diadenosine tetraphosphate in the presence of Mg2+ is 10 microM and in the presence of Zn2+ 40 microM. Adenosine 5'-tetraphosphate, guanosine 5'-tetraphosphate, and fluoride proved to be inhibitors of the diadenosine tetraphosphate hydrolase; the I50 values were 6, 10, and 20 microM, respectively. Diguanosine tetraphosphate, bis-2,6-diaminopurine beta-D-ribofuranoside tetraphosphate, and diadenosine pentaphosphate were substrates for the hydrolase; relative velocities of hydrolysis estimated for 0.5 mM diadenosine tetraphosphate and these other substrates were 1:0.51:0.44:0.20, respectively. Diadenosine tetraphosphate analogues with P2-P3 bridges such as -CF2-, -CCl2-, and -CH2- were hydrolyzed to adenosine 5'-phosphate and the corresponding adenosine 5'-triphosphate analogue.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human placental (Asymmetrical) diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase: purification to homogeneity and some properties. 838 Oct 42


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