ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication.

Simian virus 40 (SV40) large tumor antigen (T antigen) binds to two contiguous sites at the SV40 origin of replication. Of these two sites, I and II, only site II is critical for replication. We have studied the interaction between T antigen and these sites by two methods--nitrocellulose filter binding and DNase I protection. We show that T antigen binds with high occupancy to site I at 0 degrees C, 25 degrees C, and 37 degrees C but to site II only at 0 degrees C and 25 degrees C. At 37 degrees C, the temperature essential for the initiation of SV40 DNA replication in vitro, ATP is required for the interaction of T antigen and site II. ATP can be replaced efficiently by adenosine 5'-[beta,gamma-imido]triphosphate and ADP, suggesting that hydrolysis of the nucleotide is not essential for the binding of T antigen to site II. The binding to the region critical for replication can occur in the presence of a variety of nucleoside triphosphates; dATP supports binding at a concentration 1/30th that of ATP, while dGTP and rGTP were inactive at all concentrations tested.     

Nucleotide-stimulated proteolysis of histone H1.

We have identified a proteolytic system that selectively degrades histone H1 in normal human lymphocytes. Treatment of permeabilized human lymphocytes with a series of nucleotides produced a marked decrease in their histone H1 content compared to untreated cells. The nucleotide-stimulated process was selective for histone H1 because gel electrophoresis showed that almost all other lymphocyte protein bands remained constant while histone H1 disappeared. The elimination of histone H1 appears to be the result of proteolysis by a trypsin-like enzyme because it was inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor, and diisopropyl fluorophosphate. Proteolysis was stimulated by P1,P4-di(adenosine-5') tetraphosphate, P1,P3-di(adenosine-5') triphosphate, P1,P5-di(adenosine-5') pentaphosphate, adenosine 5'-tetraphosphate, ATP, adenosine 5'-[alpha, beta-methylene]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, ADP, CTP, GTP, UTP, dATP, or pyrophosphate, whereas AMP, adenosine, adenosine diphosphoribose, NAD+, cAMP, or sodium phosphate did not show this stimulation of proteolysis. ATP, [alpha, beta-methylene]ATP, [beta, gamma-methylene]ATP, and pyrophosphate all stimulated proteolysis, suggesting that a pyrophosphate linkage was necessary for this process. Thus, resting human lymphocytes contain a trypsin-like protease that is stimulated by nucleotides or pyrophosphate to selectively degrade histone H1.     

Effects of adenosine and adenine nucleotides on the atrioventricular node of isolated guinea pig hearts

The primary goal of this study was to determine whether the slowing of atrioventricular (AV) conduction by ATP is caused by ATP per se or is mediated by adenosine formed from ATP degradation. We assessed the effects of ATP, beta, gamma-methylene ATP, ADP, AMP, and adenosine on AV conduction time in the isolated perfused guinea pig heart. The cardiac effluent was collected and analyzed for its content of adenine nucleotides and nucleosides. Perfused ATP was rapidly and almost completely broken down to AMP and adenosine; only 2.5 +/- 0.5% of the infused ATP was recoverable in the effluent. A significant correlation was found between the effluent concentration of adenosine and atria-to-His bundle (A-H) conduction time. Compounds that altered the effect of adenosine on A-H conduction likewise altered the effect of ATP: (1) aminophylline, a competitive antagonist of adenosine, antagonized the ATP-induced A-H prolongation; (2) adenosine deaminase, the enzyme responsible for the deamination of adenosine to inosine, reduced the effect of ATP by 82%; (3) the adenosine transport blockers NBMPR and dipyridamole markedly enhanced the effect of ATP; and (4) EHNA, an inhibitor of adenosine deaminase, potentiated the effect of ATP. Furthermore, the less hydrolyzable ATP analog, beta, gamma-methylene ATP, was less potent than ATP in causing A-H prolongation. We conclude that the adenosine-like action of ATP on the guinea pig AV node requires that ATP first be degraded to adenosine.     

ATP-dependent regulation of cytoplasmic microtubule disassembly

Indirect immunofluorescent staining with an antitubulin antibody was used for studying the role of ATP in the regulation of cytoplasmic microtubule disassembly. Depletion of the cellular ATP pool in cultured mouse fibroblasts with various inhibitors of energy metabolism leads to inhibition of the microtubule disassembly induced by colcemid or vinblastine. Glucose added to the inhibitor-containing incubation medium partially restores the cellular ATP content and abolishes the inhibition of microtubule disassembly. The metabolic inhibitors did not change [3H]colcemid uptake by the cells; therefore, their action on the microtubule disassembly was not caused by the reduction in intracellular colcemid. Addition of ATP to the cytoskeleton preparations obtained by Triton X-100 treatment of the cells markedly stimulates microtubule depolymerization. This effect was specific for ATP; it was not observed in the presence of GTP, UTP, CTP, ADP, AMP, adenosine 5'-(beta, gamma-methylene)triphosphate (a nonhydrolyzable analogue of ATP), or inorganic pyrophosphate or tripolyphosphate. Therefore, depletion of the cellular ATP pool reduces the rate of microtubule disassembly whereas addition of ATP increases it. These results suggest that a certain ATP-dependent reaction [most probably, phosphorylation of some of the microtubule protein(s)] controls microtubule disassembly in the cells.     

甘油三酯与高密度脂蛋白预测冠状动脉病变的意义

腺苷以及ＡＭＰ、ＡＤＰ、ＡＴＰ等腺嘌呤衍生物是一类强有力的血管扩张剂。离体猪冠状动脉实验证明，ＡＴＰ、ＡＤＰ为内皮依赖性舒张作用，而腺苷本身则为非内皮依赖性舒张效应。用噻唑蓝比色法观察到与腺苷一样，ＡＭＰ、ＡＤＰ、ＡＴＰ也能剂量依赖性抑制培养的主动脉平滑肌细胞增殖。结果表明，腺苷以及ＡＭＰ、ＡＤＰ、ＡＴＰ等腺嘌呤衍生物可能是平滑肌细胞增殖的调节因子。本文还对腺苷类药物的临床应用价值进行了讨论。     

甘油三酯与高密度脂蛋白预测冠状动脉病变的意义

７８例患者选择性冠状动脉（冠脉）造影，其中冠脉病变５１例，冠脉正常２７例。测定了其高密度脂蛋白（ＨＤＬ）及其亚组分和甘油三酯（ＴＧ）。结果表明：冠脉狭窄与ＨＤＬ２负相关（ｒ＝－０．３２，Ｐ＜０．０１）、与ＴＧ正相关（ｒ＝０．３４，Ｐ＜０．０１），认为高ＴＧ和低ＨＤＬ２对于预测冠脉病变有重要意义。     

腺苷及腺嘌呤衍生物对离体猪冠状动脉环和主动脉平滑肌细胞增殖的影响

腺苷以及ＡＭＰ、ＡＤＰ、ＡＴＰ等腺嘌呤衍生物是一类强有力的血管扩张剂。离体猪冠状动脉实验证明，ＡＴＰ，ＡＤＰ为内皮依赖性舒张作用，而腺苷本身 则为非内皮依赖性舒张效应。用噻唑蓝比色法观察到与腺苷一样，ＡＭＰ、ＡＤＰ、ＡＴＰ也能剂量依赖性抑制培养的主动脉平滑肌细胞增殖。结果表明，腺苷以及ＡＭＰ、ＡＤＰ、ＡＴＰ等腺嘌呤衍生物可能是平滑肌细胞增殖的调节因子，本文还能腺苷类药物的临床应用价值进行了讨论。     

Purification and properties of the uvrA protein from Escherichia coli.

The uvrA+ gene product from Escherichia coli was purified to apparent homogeneity; the assay measured its ability to restore repair endonuclease activity in extracts from uvrA mutated cells. The uvrA protein is a 115,000 molecular weight DNA-binding protein having higher affinity for single-stranded than double-stranded DNA. It does not introduce single-strand breaks or alkali-labile bonds in native or UV-irradiated DNA, but it catalyzes hydrolysis of ATP to ADP and Pi. The ATPase activity is not DNA dependent and has a Km of 0.23 mM, which corresponds to the Km for the ATP requirement of the UV-endonuclease reaction catalyzed by the combined uvrA+, uvrB+, and uvrC+ gene products. ADP and adenosine 5'-[gamma-thio]triphosphate both inhibit the uvrA ATPase as well as the uvrABC endonuclease and also prevent specific binding of the uvrA proteins to UV-irradiated DNA. These results indicate that both the DNA-binding property and the ATPase activity of the uvrA protein are essential for uvrABC endonuclease activity and that the ATP requirement of the endonuclease reaction is determined by uvrA ATPase.     

Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin.

The proteasome (the multicatalytic endoproteinase complex) in mammalian tissues hydrolyzes proteins and several types of peptides. When this structure was isolated rapidly from rabbit skeletal muscle in the presence of glycerol, its various peptidase and protease activities showed a large reversible activation by physiological concentrations of ATP (Ka = 0.3-0.5 mM). Hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) was stimulated up to 12-fold by ATP, whereas degradation of casein and bovine serum albumin increased 4- to 7-fold. Neither ADP nor AMP had any effect. CTP, GTP, UTP, and the nonhydrolyzable analogs adenosine 5'-[beta,gamma-imino]triphosphate (AMPP[NH]P) and adenosine 5'-[alpha,beta-methylene]triphosphate (AMP[CH2]PP) increased peptide hydrolysis as well as ATP did. However, only ATP stimulated casein breakdown and only in the presence of Mg2+. Thus, nucleotide binding allows activation of the peptidase functions, but ATP hydrolysis seems necessary for enhanced degradation of proteins. The ATP effect on proteolysis was reversible and did not require ubiquitin. Sensitivity to ATP was labile, and with storage at 4 degrees C the enzyme became fully active in the absence of ATP or Mg2+. The ATP-activated form closely resembles the proteasome complex described previously, which did not show ATP dependence: both have molecular masses of 650 kDa, contain the same 8-10 subunits, and are precipitated by the same antibodies. A similar ATP-activated form was found in rabbit liver but not in rabbit reticulocytes. The proteasome seems to represent a ubiquitin-independent, ATP-stimulated proteolytic activity within nucleated mammalian cells.     

In vitro assembly of the Bacillus subtilis bacteriophage phi 29.

In vitro assembly of the Bacillus subtilis bacteriophage phi 29 that approaches the efficiency of assembly in vivo has been demonstrated. Proheads, DNA, and gene 16 product (gp16) were essential for DNA encapsidation, and the average yield in extracts was 180 phage per prohead donor cell. The in vitro maturation was very similar to in vivo assembly in terms of yield, intermediates, and abortive structures. More that 30% of the proheads in the extract were converted to phage, and about 20% of DNA--protein extracted from phage could be repackaged. In vitro assembly was blocked by the addition of DNase I, EDTA, pyrophosphatase, or the ATP analogues adenosine 5'-[alpha, beta-methylene]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate. Less than 1% of the proheads isolated in sucrose gradients can accept DNA--protein in packaging in vitro.     

Effects of adenine nucleotides on the proliferation of aortic endothelial cells.

The effects of adenine nucleotides and adenosine on DNA synthesis and cell growth have been studied in bovine aortic endothelial cells (BAECs). ATP produced a small but significant (+44%) increase of the fraction of BAECs whose nuclei are labeled by [3H]thymidine. This mitogenic effect was mimicked by ADP, the phosphorothioate analogues ATP gamma S and ADP beta S, and the nonhydrolyzable analogue adenosine 5'-(beta, gamma-imido)triphosphate (APPNP), whereas adenosine 5'-(alpha, beta-methylene)triphosphate (APCPP), a selective agonist of P2x-purinoceptors, had no effect at 10 microM and a small one at 100 microM; this profile is consistent with the involvement of P2y-receptors. Adenosine induced a mitogenic response of a magnitude similar to that of ATP. This effect was not reproduced by R-phenylisopropyl adenosine, by 5'-N-ethylcarboxamide adenosine, or by 2',5'-dideoxyadenosine, selective ligands of the A1- and A2-receptors and the P site, respectively, nor was it inhibited by 8-phenyltheophylline, an antagonist of both A1- and A2-receptors. The mechanism of this adenosine action thus remains unclear. ATP and ATP gamma S did not enhance the proliferation of BAECs cultured in the presence of fetal calf serum concentrations ranging from 0.5% to 10%. They inhibited the growth-promoting effect of basic fibroblast growth factor; among the various nucleotides tested, APCPP was the least effective to reproduce the action of ATP, suggesting the possible involvement of P2y-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.

The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[gamma-thio]triphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coli.     

Potassium conductance of the squid giant axon is modulated by ATP.

This communication reports a modulating effect of intracellular ATP on the steady-state and kinetic properties of the delayed rectifier of the giant axon of the squid. When internally dialyzed or perfused giant axons from Loligo plei or Loligo pealei are voltage clamped at -60 mV and washed free of ATP, the potassium current at 0 mV is decreased, and the time course of the potassium current is faster. Upon readmitting ATP, the potassium current for pulses to potentials more positive than -30 mV is increased by a factor of up to 2.5, while for pulses to potentials more negative than -30 mV, it is decreased. In the presence of ATP the turn-on of the time course of the potassium current is slower, but the turn-off of the time course is faster. The effect of ATP is only observed when magnesium ions are present in the internal medium; ADP or the nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-methylene]-triphosphate has no effect. When the holding potential is -70 mV, the conductance-voltage curve is shifted to more positive potentials by ATP, but the maximum conductance is only slightly increased. Most of the effects of ATP may be explained by a phosphorylation step that alters the voltage sensor of the activation and inactivation gates of the potassium channels shifting the voltage dependence of both processes to more depolarized potentials.     

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.     

recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.

The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo.     

Cartilage nucleoside triphosphate pyrophosphohydrolase. II. Role in extracellular pyrophosphate generation and nucleotide metabolism

Extracellular generation of inorganic pyrophosphate (PPi) in cartilage organ culture is markedly augmented by ATP.ATP, not an ATP metabolite (ADP, AMP, adenosine) is necessary for this augmentation. Excess PPi production is effectively blocked by known inhibitors of nucleoside triphosphate (NTP) pyrophosphohydrolase (EDTA, EGTA, dithiothreitol). Excess 32P-PPi is generated directly from gamma 32P-ATP by cartilage, as substrate and product have similar specific activities. These findings strongly favor ecto-NTP pyrophosphohydrolase as the source of extracellular PPi generation in the presence of NTP. Additionally, active nucleotide and nucleoside catabolism is demonstrated in these cartilage organ cultures.     

Responses to adenine nucleotides and related compounds of isolated dog cerebral, coronary and mesenteric arteries

In helically cut strips of dog cerebral, coronary and mesenteric arteries contracted with prostaglandin F2 alpha, adenosine, AMP, ADP, ATP and cyclic AMP produced dose-related, persistent relaxation. On the basis of ED50S, the relaxant effect seen in cerebral arteries was in the order of AMP, ADP, ATP greater than adenosine, cyclic AMP greater than aderine, dibutyryl cyclic AMP greater than inosine. On the basis of maximum relaxations, the effect was in the order of adenine, dibutyryl cyclic AMP greater than or equal to adenosine, AMP, ADP, ATP greater than cyclic AMP, inosine. The relaxant responses to adenosine, AMP, ADP, ATP and cyclic AMP were attenuated by treatment with aminophylline, whereas the relaxations induced by dibutyryl cyclic AMP, adenine and inosine were not altered. It is concluded that adenosine, AMP, ADP and ATP, but not adenine and inosine, appear to share receptors underlying arterial relaxations, and the cyclic AMP-induced relaxation does not derive from increments in cellular cyclic AMP. Treatment with aspirin did not alter the relaxant effect of ADP, adenosine and adenine, suggesting that the release of vasoactive prostaglandins is not involved.     

Changes in gastric mucosal content of adenosine, xanthine and hypoxanthine induced by restrained water immersion stress: antiulcer effects of tetraprenylacetone

The purpose of this study was to clarify the involvement of adenine nucleotide metabolism and substrates of the xanthine-xanthine oxidase system as the source of oxygen free radicals in a rat model of restrained water immersion stress ulceration. The gastric mucosal concentrations of adenine-5'-triphosphate (ATP), adenine-5'-diphosphate (ADP), adenine-5'-monophosphate (AMP) and thiobarbituric-acid (TBA)-reactant substances were measured after 4, 8 and 12 h restrained water immersion stress. The gastric mucosal concentrations of the nucleoside adenosine, the purine bases xanthine and hypoxanthine, and the final metabolic product uric acid, were measured after 4 h of restrained water immersion stress. The concentrations of ATP diminished significantly after 4, 8 and 12 h of restrained water immersion stress. However, the observed stress-induced changes in ADP were not significant. AMP concentrations increased significantly after 4, 8 and 12 h of stress. The adenylate pool (ATP + ADP + AMP) dropped significantly from the prestress value after 4, 8 and 12 h of stress, and the concomitant energy charge (EC = ATP + 0.5 ADP/ATP + ADP + AMP) decreased significantly after 4 and 8 h of stress compared with the prestress value. Gastric mucosal concentrations of TBA-reactant substances displayed a significant increase after 4 h of stress, and remained unchanged after 8 and 12 h of stress from the level after 4 h. Four hours of restrained water immersion stress induced an increase in adenosine and uric acid concentrations and a decrease in the hypoxanthine concentration of the gastric mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP and other nucleoside triphosphates inhibit the binding of insulin to its receptor

ATP, in a dose-dependent manner, inhibited the binding of 125I-insulin to its receptor in rat liver and human placental membranes. With rat liver plasma membranes an effect of ATP was detected at concentrations between 1.0 and 2.5 mmol/L, and maximal effects were seen at 10.0 mmol/L where binding was decreased by approximately 40%. The effect of ATP was one half-maximal within 10 minutes and maximal within 60 minutes. Scatchard analysis indicated that ATP was acting primarily to change the binding affinity of the insulin receptor. The effect of ATP was mimicked by CTP, GTP, and UTP, but not by ADP, 5'-AMP, 3'-AMP, 3'5'-cyclic AMP and adenosine. The ATP analog AMP-PNP had a potency approximately 10% that of ATP. The effect of ATP was not significantly influenced by inhibitors of phosphoprotein kinases and phosphoprotein phosphatases. In human placental membranes, ATP had a similar effect in inhibiting 125I-insulin binding to its receptor. Moreover, ATP was active in inhibiting insulin binding to purified human placental insulin receptors at 0.01 mmol/L, a concentration 1/100 of that needed for inhibiting binding to intact membranes. These studies indicate, therefore, that ATP and other nucleoside triphosphates influence the ability of the insulin receptor to bind insulin.