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1.
The hydrolytic activity of mitochondrial ATPase, both in its soluble form as F1-ATPase, or as membrane bound in whole mitochondria, was affected by the presence of free nucleoside di- or triphosphates; these effects were largely depending not only on their concentration but also on the substrate concentration. The existence of a regulatory site or sites is proposed; these sites would have a higher affinity for the free nucleoside triphosphates than for the diphosphates, and the interaction of any of these nucleotides with the proposed regulatory site or sites would lead to an activation. The nucleotide regulatory site or sites seem to be different from the anion binding sites since neither free ATP nor free GTP compete with activating or inhibitory anions. 相似文献
2.
Evidence that Mg2+- or Ca2+-activated adenosine triphosphatase in rat pancreas is a plasma-membrane ecto-enzyme. 总被引:1,自引:1,他引:0
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Preparations of enzymically dispersed rat pancreatic cells hydrolyse externally added nucleoside triphosphates and diphosphates at high rates in the presence of Mg2+ or Ca2+. The lack of response to specific inhibitors and activators differentiates this hydrolytic activity from that of other well-characterized ion-transporting ATPases. Studies based on inactivation of this hydrolytic activity by the covalently reacting, slowly permeating probe diazotized sulphanilic acid indicated that this nucleoside tri- and di-phosphatase is primarily a plasma-membrane ecto-enzyme. It is the major ATPase activity associated with intact cells, homogenates and isolated plasma-membrane fractions. Concanavalin A stimulates this ATPase activity of intact cells and isolated plasma-membrane fractions. The insensitivity of this ATPase activity to univalent ions and inhibitors of pancreatic electrolyte secretion, taken together with the evidence that the active site is externally located, suggests that this enzyme is not directly involved in HCO3- secretion in the pancreas. Its actual function remains unknown. 相似文献
3.
The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP. 总被引:48,自引:0,他引:48
A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [(32)P]AMP, and cyclic [(32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of [gamma-(32)P]ATP, [gamma-(32)P]GTP, [gamma-(32)P]ITP, and [gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various [alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with [gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5'-(32)P]nucleoside monophosphates. The [5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [(32)P]AMP and cyclic [(32)P]GMP are also prepared enzymatically from [alpha-(32)P]ATP or [alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for [gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [alpha-(32)P]nucleoside triphosphates. Methods for the use of the [alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the [alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems. 相似文献
4.
A simple, rapid, and inexpensive method is described for the synthesis of γ-32P-labeled ribo- or deoxyribonucleoside triphosphates. The procedure involves chemical synthesis of [32P]acetylphosphate and subsequent phosphorylation of nucleoside diphosphates using acetate kinase (EC 2.7.2.1) and a final purification step. The entire procedure is performed 8 h or less. 相似文献
5.
With appropriate preparations of spinish chloroplasts we observe three distinct effects of the nucleotides: 1. An accelaration of the dark decay of the light induced 520 nm absorbance change after ATP addition. 2. An acidification of the internal space of the thylakoids after ATP addition in darkness. 3. A dark ATPase activity which is regulated by the deltapH across the membrane. We conclude that the effect of the nucleoside triphosphates on the 520 nm signal is linked to a change of the proton conductivity of the membrane, induced by the formation of a deltapH across the membrane in consequence of the dark ATPase activity. The mode of action of the nucleoside diphosphates in the presence of inorganic phosphate on the 520 nm signal is discussed. It is proposed that the effects observed are linked to the hydrolysis of the newly formed nucleoside triphosphates. 相似文献
6.
7.
We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3′ single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The kcat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3′ stem-loop structure, and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3′ stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 μM ADP, CDP, GDP, and UDP, the Km for the phosphorolysis of the substrate with the 3′ stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme. 相似文献
8.
Two forms of DNA-dependent ATPase activity have been purified from yeast extracts. The two ATPases differ from each other in chromatographic properties and heat stabilities but have similar molecular weight and reaction properties. DNA-dependent ATPase I has been purified to near homogeneity, while DNA-dependent ATPase II is only partially purified. The two ATPases from yeast are related structurally since antiserum raised against ATPase I cross-react against ATPase II. Yeast DNA-dependent ATPase I has a native molecular weight of about 68,000 and consists of a single polypeptide chain. ATPase II also sediments on sucrose gradient as a 68,000-dalton protein. Both yeast DNA-dependent ATPases hydrolyze dNTPs and rNTPs to their corresponding nucleoside diphosphates and orthophosphate, but dATP and ATP are preferred substrates. In addition to nucleoside triphosphates, both enzymes require a divalent cation and a polynucleotide for activity. Single-stranded DNAs and polydeoxynucleotides are the most effective co-substrates for yeast DNA-dependent ATPases. Addition of yeast DNA-dependent ATPases to DNA synthesis system containing yeast DNA polymerases does not significantly stimulate the rate of DNA synthesis. 相似文献
9.
Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli. 总被引:1,自引:0,他引:1
N K Avliyakulov J Lukes A V Kajava B Liedberg I Lundstr?m S P Svensson 《European journal of biochemistry》2000,267(6):1723-1731
Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity. In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro. L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe. The cloned gene was overexpressed and purified as a his-tag fusion protein in E. coli. Radioligand binding experiments, using [gamma-35S]ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity. Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates. Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues. Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of [gamma-35S]ATP at subclinical levels. Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts. 相似文献
10.
Ribonucleotide reductases (RNRs) are required for the synthesis of deoxyribonucleoside triphosphates (dNTPs) from ribonucleotides. In Escherichia coli, regulation of RNR expression is co‐ordinated with the cell cycle, and involves several regulatory proteins. One of these, NrdR, has recently been shown to regulate all three nrd operons that encode RNR isoenzymes. Repression by NrdR is believed to be stimulated by elevated dNTPs, although there is no direct evidence for this model. Here, we sought to elucidate the mechanism by which NrdR regulates nrd expression according to the abundance of (d)NTPs. We determined that ATP and dATP bind to NrdR in a negatively cooperative fashion, such that neither can fully occupy the protein. Both nucleotides also appear to act as positive heterotropic effectors, since the binding of one stimulates binding of the other. Nucleotide binding stimulates self‐association of NrdR, with tri‐ and diphosphates stimulating oligomerization more effectively than monophosphates. As‐prepared NrdR contains (deoxy)nucleoside monophosphates, diphosphates and triphosphates, and its DNA binding activity is inhibited by triphosphates and diphosphates but not by monophosphates. We propose a model in which NrdR selectively binds (deoxy)nucleoside triphosphates, which are hydrolysed to their monophosphate counterparts in order to regulate DNA binding. 相似文献
11.
An ATPase was purified from mouse myeloma MOPC 70E the activity of which depends on the presence of single-stranded DNA and divalent cations such as Mg2+, Mn2+, Ca2+, Ni2+ or Fe2+. The enzyme splits both ribonucleoside and deoxyribonucleoside triphosphates but preferentially ATP and dATP yielding nucleoside diphosphates and inorganic phosphate. The enzyme has an absolute requirement for single-stranded DNA. Alternating double-stranded polydeoxynucleotides are only slight effective, and native double-stranded DNA, single-stranded and double-stranded RNAs as well as DNA - RNA hybrids are ineffective in stimulating the ATPase. The enzyme has further characterized by sedimentation in a sucrose density gradient (s20, w = 5.5 S) and by isoelectric focussing in an ampholine pH gradient (pI = 6.5). 相似文献
12.
A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes. 相似文献
13.
Allosteric activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by nucleoside diphosphates 总被引:3,自引:0,他引:3
Extensively purified rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase was used to examine the role of ADP in inactivation of HMG-CoA reductase (EC 1.1.1.34). Solubilized HMG-CoA reductase was a suitable substrate for HMG-CoA reductase kinase. At sufficiently high concentrations of solubilized HMG-CoA reductase, reductase kinase activity approached that measured using microsomal HMG-CoA reductase as substrate. Inactivation of solubilized HMG-CoA reductase by HMG-CoA reductase kinase required both MgATP and ADP. Other nucleoside diphosphates, including alpha, beta-methylene-ADP, could replace ADP. HMG-CoA reductase kinase catalyzed phosphorylation of bovine serum albumin fraction V by [gamma-32P]ATP. This process also required a nucleoside diphosphate (e.g. alpha, beta-methylene-ADP). Nucleoside diphosphates thus act on HMG-CoA reductase kinase, not on HMG-CoA reductase. For inactivation of HMG-CoA reductase, the ability of nucleoside triphosphates to replace ATP decreased in the order ATP greater than dATP greater than GTP greater than ITP, UTP. TTP and CTP did not replace ATP. Both for inactivation of HMG-CoA reductase and for phosphorylation of bovine serum albumin protein, the ability of nucleoside diphosphates to replace ADP decreased in the order ADP greater than CDP, dADP greater than UDP. GDP did not replace ADP. Nucleoside di- and triphosphates thus appear to bind to different sites on HMG-CoA reductase kinase. Nucleoside diphosphates act as allosteric activators of HMG-CoA reductase kinase. For inactivation of HMG-CoA reductase by HMG-CoA reductase kinase, Km for ATP was 140 microM and the activation constant, Ka, for ADP was 1.4 mM. The concentration of ADP required to modulate reductase kinase activity in vitro falls within the physiological range. Modulation of HMG-CoA reductase kinase activity, and hence of HMG-CoA reductase activity, by changes in intracellular ADP concentrations thus may represent a control mechanism of potential physiological significance. 相似文献
14.
Marcella Camici Carlo Fini Pier Luigi Ipata 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,840(1):6-12
5′-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5′-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg2+. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The Ki values for ADP and ATP are 0.036 and 0.28 μM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation. 相似文献
15.
Del Favero M Mazzantini E Briani F Zangrossi S Tortora P Dehò G 《The Journal of biological chemistry》2008,283(41):27355-27359
Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase. 相似文献
16.
Summary The strong enzyme histochemical reactions for adenosine triphosphatase (ATPase) seen in ependymal tanycytes after incubation in calcium-containing media have previously been reported as calcium transport ATPase. Investigation of these reactions showed that: (1) any nucleoside triphosphate can serve as a substrate; (2) diphosphates and monophosphates cannot replace triphosphates; this includes p-nitrophenyl phosphate which is readily hydrolysed by plasma membrane transport ATPases; (3) strong localization occurs in the presence of millimolar concentrations of either calcium or magnesium ions; there is no absolute requirement for calcium ions; (4) they are not inhibited by sulphydryl inhibitors or calmodulin antagonists; (5) lead phosphate precipitates are localized almost entirely on the external face of tanycyte plasma membranes. In addition, the technique gives strong localization to vessels in the choroid plexus but not to the choroidal epithelium. Immunohistochemistry with a primary antibody raised against Ca2+,Mg2+-ATPase stains the choroidal epithelium but not the vessels or the ependymal tanycytes. These results are inconsistent with identification of the reaction as calcium transport ATPase but support characterization as an ecto-ATPase. 相似文献
17.
Effect of Nucleotides and Other Inhibitors on the Inactivation of Glutamate Decarboxylase 总被引:5,自引:4,他引:1
Abstract: The effects of 17 nucleotides and nucleotide analogs and 11 other compounds on the glutamate-promoted inactivation of brain glutamate decarboxylase were examined. Among the nucleotides, the major determinant of potency was the polyphosphate chain, Glutamate-promoted inactivation was strongly enhanced by low concentrations (<100 μM) of adenosine tetraphosphate and all eight nucleoside triphosphates tested. Nucleoside diphosphates enhanced inactivation, but were much less effective than the nucleoside triphosphates; nucleoside monophosphates were not effective. Modification of the polyphosphate chain of the nucleoside triphosphates also affected potency; adenylylimidodiphosphate and α,β-methylene ATP were about as effective as nucleoside diphosphates, but α,β-methylene ATP was nearly as effective as ATP. The nucleoside base had only a small effect on potency; purine nucleotides were more potent than pyrimidine nucleotides, and one nucleotide with a tricyclic base, 1, N6 -etheno ATP, was as effective as the purine nucleoside triphosphates. The 2'-hydroxyl group of ribose was unimportant, since deoxy ATP was as effective as ATP. Three nonnucleotide polyanions were strong promoters of inactivation; inositol hexasulfate and 5-phosphorylribose 1-pyrophosphate were at least as effective as ATP; inositol hexaphosphate (phytate) was as effective as the nucleoside diphosphates. These results suggest that a major determinant of potency was a strong negative charge on the molecule. Negative charge was not sufficient, however, since fructose 1,6-bisphosphate did not promote inactivation. Inactivation by all of these compounds was slow, requiring more than 20 min for full effect. Two competitive inhibitors, chloride and glutarate, acted immediately and also reduced rather than enhanced glutamate-promoted inactivation. 相似文献
18.
Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme. 相似文献
19.
2-Methylthioadenosine 5'-mono- and diphosphates were prepared by chemical synthesis, and enzymatically phosphorylated to 5'-[beta-32P]-diphosphate and 5'-[gamma-32P]-triphosphate, respectively. They were isolated by HPLC and had activity exceeding 1000 Ci/mmol and radiochemical purity higher than 95%. 相似文献
20.
Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase activity is enhanced about 5 fold by 2 mM of either AMP or ADP. Activation constants, Ka, for AMP and ADP are 17 microM and 430 microM respectively, showing that AMP is a more potent activator than ADP. This property is expressed by increasing not only the rate of reductase inactivation but also the rate of reductase phosphorylation from [gamma-32P]ATP. GTP can replace ATP as substrate of reductase kinase but GMP and GDP cannot replace AMP as activators. Kinetic studies show that ATP can only act as a substrate. Nucleoside mono or diphosphates and nucleoside triphosphates, thus, appear to bind to different sites on microsomal HMG-CoA reductase kinase. Nucleoside mono or diphosphates act as allosteric activators of reductase kinase. The adenosyl moiety and the unaltered phosphate ester at the 5' position are two essential features of the activator molecule. Phosphorylation of reductase either by microsomal or cytosolic AMP-activated reductase kinase produces an 80% inactivation, with a concomitant incorporation of 0.8 mol of 32P per mol of reductase (Mr 55,000). In both cases exhaustive tryptic digestion of 32P-labeled HMG-CoA reductase, which had been denatured in 2M urea, yields two major phosphopeptides, the phosphoryl group being bound to serine residues. 相似文献