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

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

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

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

The substrate specificity of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum for dinucleoside polyphosphates has been determined by high-performance liquid chromatography (HP-LC). Elution of a strong anion-exchange resin with a pH and ionic strength gradient of ammonium phosphate separates a series of monoadenosine and diadenosine polyphosphates. Most of the corresponding guanine nucleotides are also resolved on this HPLC system. One mole each of Ap4A and Gp4G is symmetrically hydrolyzed to 2 mol of ADP and GDP, respectively. Ap3A, Ap5A, Ap6A, and Ap4 are hydrolyzed, and in each case ADP is one of the products. Gp3G, Gp5G, Gp6G, and Gp4 are also substrates, and in each case GDP is one of the products. AMP, ADP, ATP, Ap2A, ADPR, GMP, GDP, GTP, NAD+, and NADP+ are not substrates. No hydrolysis of the cap dinucleotides m7Gp3Am and m7Gp3Cm was detected by HPLC. Diadenosine tetraphosphate pyrophosphohydrolase preparations were also assayed for adenylate kinase, nucleotide diphosphate kinase, NAD(P)+ pyrophosphohydrolase, phosphodiesterase, cyclic nucleotide phosphodiesterase, phosphatase, and ribonuclease activities. These enzymic activities were not detectable in diadenosine tetraphosphate pyrophosphohydrolase. The symmetrical hydrolysis of Ap4A and Gp4G is an unique catalytic property that distinguishes diadenosine tetraphosphate pyrophosphohydrolase from P. polycephalum from diadenosine tetraphosphate phosphohydrolases from other organisms.
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PMID:Diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum. Substrate specificity. 629 57

Diadenosine tetraphosphatase, an enzyme splitting diadenosine tetraphosphate to AMP and ATP, has been purified to apparent homogeneity from a permanent cell line derived from a leukemic child. The purification procedure consisted of fractionation by ammonium sulfate precipitation, followed by Sephacryl 200 and DEAE-cellulose chromatography, and finally a differential membrane filtration. The enzyme is a single polypeptide chain of Mr = 17,500 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent molecular weight of the native enzyme was calculated as 20,000 from gel filtration data. The apparent Km for Ap4A was 0.5 microM as determined by two independent kinetic assays. None of the following compounds were substrates of the enzyme: diadenosine triphosphate, NAD, nucleoside 5'-phosphates (AMP, ATP, GDP, GTP, and UTP). The enzyme had optimal activity in the presence of 1 mM Mg2+, showing no activity in the presence of EDTA.
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PMID:Diadenosine tetraphosphatase from human leukemia cells. Purification to homogeneity and partial characterization. 630 76

Interactions of eukaryotic 5-dimethylaminonaphthalene-1-sulfonyl-initiation factor 2 (eIF-2) from rabbit reticulocytes and the guanine nucleotide exchange factor ( GEF ), Met-tRNAf, GTP, and GDP were monitored by changes in fluorescence anisotropy and radioactive filtration assays. At 1 mM Mg2+, radioactive filtration assays demonstrate that GEF is necessary for nucleotide exchange. We did not observe a GDP dependence in the association reaction of eIF-2 X GEF for GDP concentrations from 0.01 to 20 microM. This is in disagreement with the model: eIF-2 X GDP + GEF in equilibrium eIF-2 X GEF + GDP. The addition of GTP caused a decrease in fluorescence anisotropy which is interpreted as a dissociation of eIF-2 X GEF . We propose an asymmetrical model of ternary complex (eIF-2 X GTP X Met-tRNAf) formation where 1) GDP does not displace GEF and 2) GTP replaces GEF and presumably GDP. For reticulocyte eIF-2, phosphorylation of the alpha subunit greatly inhibits protein synthesis. This inhibition derives neither from failure of GEF to bind to eIF-2(alpha P) nor from greatly enhanced binding of GEF . The inhibition results from the requirement of very high levels of GTP (100 microM) to dissociate the eIF-2(alpha P) X GEF complex.
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PMID:Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation. 633 54

The present study describes the subcellular distribution of low molecular weight GTP-binding proteins in the human carcinoma cell line Caco-2. Highly enriched subcellular fractions of basolateral and brush border membranes were prepared by differential density centrifugation and divalent cation precipitation. Small molecular weight GTP-binding proteins were identified after SDS-PAGE transfer to nitrocellulose using [alpha 32P]-labelled GTP. Smg-proteins with molecular masses of 28, 27, 25 and 24 kDa were detectable in all fractions. Homogenate and brush border membrane fraction showed specific binding of [alpha 32P] GTP to proteins of a molecular mass of 21 kDa, while in the membrane fractions (apical, basolateral) a high enrichment of 24 kDa smg-proteins was detectable. Western blot analysis identified one of the 21 kDa proteins of the brush border membrane as rhoA. The homogenate of 4, 8, 11 and 14 days old Caco-2 cells showed different [alpha 32P]-GTP binding to 21, 27 and 28 kDa proteins. In conclusion, this study is the first showing the presence and asymmetrical distribution of smg-proteins among the various membrane components in the human carcinoma cell line Caco-2.
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PMID:Subcellular distribution of small GTP-binding proteins in the intestinal cell line Caco-2. 855 67

The maternal transcript of the anterior segmentation gene bicoid (bcd) is localized at the anterior pole of the Drosophila egg and translated to form a gradient in the nuclei of the syncytial blastoderm embryo after fertilization [1-3]. The nuclear gradient of Bcd protein - a transcription factor - leads to differential expression of zygotic segmentation genes. The rapid nuclear division during this stage [4] requires that Bcd quickly enters the nuclei after each mitosis using an active nuclear import system. Nuclear transport depends on the asymmetrical distribution of two forms of the small GTPase Ran: Ran-GTP is concentrated in the nucleus and Ran-GDP in the cytoplasm [5-8]. Ran requires RanGTPase-activating protein-1 (RanGAP1) on the cytoplasmic side of nuclear pore complexes to convert Ran-GTP to Ran-GDP. In vitro studies with vertebrate proteins demonstrate that the RanGAP1 associated with the nuclear pore complex is modified with small ubiquitin related modifier-1 (SUMO-1) by a ubiquitin-conjugating enzyme (E2 enzyme) [9-15]. Here, we show that mutation of the Drosophila semushi (semi) gene, which encodes an E2 enzyme, blocks nuclear import of Bcd during early embryogenesis and results in misregulation of the segmentation genes that are Bcd targets. Consequently, semi embryos have multiple defects in anterior segmentation. This study demonstrates that an E2 enzyme is required for nuclear transport during Drosophila embryogenesis.
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PMID:The Drosophila semushi mutation blocks nuclear import of bicoid during embryogenesis. 982 80

A number of eukaryotic proteins are already known to orchestrate key steps of mRNA metabolism and translation via interactions with the 5' m7GpppN cap. We have characterized a new type of histidine triad (HIT) motif protein (Nhm1) that co-purifies with the cap-binding complex eIF4F of Schizosaccharomyces pombe. Nhm1 is an RNA-binding protein that binds to m7GTP-Sepharose, albeit with lower specificity and affinity for methylated GTP than is typical for the cap-binding protein known as eukaryotic initiation factor 4E. Sequence searches have revealed that proteins with strong sequence similarity over all regions of the new protein exist in a wide range of eukaryotes, yet none has been characterized up to now. However, other proteins that share specific motifs with Nhm1 include the human Fhit tumour suppressor protein and the diadenosine 5', 5"'-P1, P4-tetraphosphate asymmetrical hydrolase of S. pombe. Our experimental work also reveals that Nhm1 inhibits translation in a cell-free extract prepared from S. pombe, and that it is therefore a putative translational modulator. On the other hand, purified Nhm1 manifests mRNA decapping activity, yet is physically distinct from the Saccharomyces cerevisiae decapping enzyme Dcp1. Moreover, fluorescence and immunofluorescence microscopy show that Nhm1 is predominantly, although not exclusively, nuclear. We conclude that Nhm1 has evolved as a special branch of the HIT motif superfamily that has the potential to influence both the metabolism and the translation of mRNA, and that its presence in S. pombe suggests the utilization of a novel decapping pathway.
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PMID:A nuclear protein in Schizosaccharomyces pombe with homology to the human tumour suppressor Fhit has decapping activity. 1236 30

Mammalian meiotic divisions are asymmetrical and generate a large oocyte and two small polar bodies. This asymmetry results from the anchoring of the meiotic spindle to the oocyte cortex and subsequent cortical reorganization, but the mechanisms involved are poorly understood. We investigated the role of Rac in oocyte meiosis by using a fluorescent reporter for Rac-GTP. We find that Rac-GTP is polarized in the cortex overlying the meiotic spindle. Polarization of Rac activation occurs during spindle migration and is promoted by the proximity of chromatin to the cortex. Inhibition of Rac during oocyte maturation caused a permanent block at prometaphase I and spindle elongation. In metaphase II-arrested oocytes, Rac inhibition caused the spindle to detach from the cortex and prevented polar body emission after activation. These results demonstrate that Rac-GTP plays a major role in oocyte meiosis, via the regulation of spindle stability and anchoring to the cortex.
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PMID:Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes. 1727 47


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