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1.
An existing enzymic method for preparing [γ-32P]ATP from 32Pi has been modified toyield [γ-32P]ATP free of salt and buffer. 32P is incorporated into the γ-position of ATP by isotopic exchange in the presence of glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase. Unreacted 32Pi is separated from [γ-32P]ATP by column chromatography on Dowex 1 bicarbonate. [γ-32P]ATP is eluted with 2 m triethylammonium bicarbonate, which is then completely removed by freeze-drying. 相似文献
2.
(i) A new, rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts in the presence of other 32P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of 32P into protein from [γ-32P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) 32Pi uptake in perfused rat heart was found to be a relatively slow process, with a Km of 0.084 mm, whereas equilibration between intracellular 32Pi and [γ-32P]ATP occurred rapidly. 相似文献
3.
A satisfactory method for the determination of the specific activity of highly labeled [γ-32P]ATP has not been reported previously. Yields of high specific activity 32P labeled material usually are too small to be detected by ultraviolet spectrophotometry or phosphate analysis. Recent reports describing the assay of ATP by enzyme catalyzed phosphate transfer to 3H labeled glucose (1) or galactose (2) are not suitable for use with highly labeled 32P material since the crossover into the 3H channel will greatly exceed the radioactivity of the 3H labeled phosphate acceptor. Recently Schendel and Wells reported the preparation of essentially carrier free [γ-32P]ATP. They indicated, however, that the specific activity of the labeled product could not be determined by conventional methods (3). We have developed and now routinely use an expedient method for the determination of the specific activity of picomole quantities of highly labeled [γ-32P]ATP. This procedure measures the phosphate transfer from [γ-32P]ATP to oligothymidylic acid [dT(pT)10] catalyzed by bacteriophage T4 induced polynucleotide kinase. The specific activity is determined by measuring the radioactivity present in d-32pT(pT)10, and can be verified by an isotope dilution method employing the same assay. Specific activities as high as 240 Ci/mmole have been determined. 相似文献
4.
A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts containing other 32P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of 32P into casein from [γ-32P]ATP is measured by perchloric acid precipitation of the protein on filter paper. 32P-Casein formation is linearly related to the specific radioactivity of the [γ-32P]ATP. Separation of ATP from other 32P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-32P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to 32Pi is demonstrated. 相似文献
5.
Jonathan R. Seals Jay M. McDonald David Bruns Leonard Jarett 《Analytical biochemistry》1978,90(2):785-795
A manual ATPase assay which measures the release of 32Pi from [γ-32P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate 32Pi from [γ-32P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10−15 to 10−7 mol of 32Pi from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-32P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay. 相似文献
6.
An enzymatic method for [32P]phosphoenolpyruvate synthesis 总被引:7,自引:0,他引:7
A convenient method for the enzymatic synthesis of [32P]phosphoenolpyruvate from [γ-32P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-32P]ATP to [32P]phosphoenolpyruvate, which was subsequently separated from residual [γ-32P]ATP and by chromatography on AG-1-X8-bicarbonate resin. 相似文献
7.
A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts containing other 32P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of 32P into casein from [γ-32P]ATP is measured by perchloric acid precipitation of the protein on filter paper. 32P-Casein formation is linearly related to the specific radioactivity of the [γ-32P]ATP. Separation of ATP from other 32P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-32P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to 32Pi is demonstrated. 相似文献
8.
A new method has been developed for prelabeling tissue ATP pools with 32P inorganic phosphate (Pi) and for the subsequent isolation of [32P]cAMP and [32P]ATP. The new method of prelabeling eliminates the need to separate trace amounts of radioactive cAMP from radioactive breakdown products of adenine formed in tissues prelabeled with [3H]- or [14C]adenine. The effect of epinephrine to increase [32P]cAMP levels in rat ventral prostate tissue fragments has been studied in terms of increase in the ratio of [32P]cAMP/[32P]ATP in the absence and presence of various phosphodiesterase inhibitors. Tissue prelabeling with 32Pi labels GTP as well as ATP (and other nucleoside triphosphates); thus the method lends itself to the isolation of [32P]cGMP as well as [32P]cAMP from the same tissue sample. 相似文献
9.
After conversion of unlabeled DNA and RNA to 3′-mononucleotides accurate base compositional analysis can be performed on as little as 10 ng of the hydrolysate. The 3′-mononucleotides are first quantitatively postlabeled with [γ-32P]ATP by T4 polynucleotide kinase and are then separated as mononucleoside diphosphates on Whatman DE-81 ion-exchange paper at pH 3.5 after hydrolysis of surplus [γ-32P]ATP to 32P1. The locations of the four labeled nucleoside diphosphates are determined by autoradiography and the ratio of radioactivity in the four spots gives the base ratio of the sample. 相似文献
10.
The specific radioactivity of the γ-phosphorus of ATP has been determined by an indirect method. Galactokinase is employed to transfer the terminal phosphate group of [γ-32P] ATP to [1-3H] galactose. The doubly labeled galactose-1-phosphate is purified by ion exchange chromatography on QAE Sephadex. The specific radioactivity of the phosphorus is calculated from the ratio. The method is extremely sensitive, requiring only 0.005 μmoles of ATP with a specific radioactivity of 1 μCi/μmole, and the chromatographic isolation of galactose-1-phosphate is simple and reproducible. The method is directly applicable to the determination of the specific radioactivity of [γ-32P] ATP in biological samples. 相似文献
11.
Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells 总被引:1,自引:1,他引:0
Tai-An Lin Kevin D. Lustig Michael G. Sportiello Gary A. Weisman Grace Y. Sun 《Journal of neurochemistry》1993,60(3):1115-1125
Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis off [32P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4-) in the medium and not with the concentration of magnesium-ATP (MgATP2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]Pi or [14C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D. 相似文献
12.
We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little 32Pi, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [3H]uridine before the experiment provides a measure of ribosome yield. The amount of 32Pi incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [32P]ATP increases, when the cells are stimulated by prostaglandin F2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the 32Pi incorporated into S6 increases by a factor greater than the increase in the specific activity of [32P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus. 相似文献
13.
Roger A. Johnson Ilana Shoshani Carmen Dessauer Gilles Gosselin 《Nucleosides, nucleotides & nucleic acids》2013,32(4-5):839-842
Abstract An enzymatic method was developed for the preparation of unlabeled and [β-32P]-labeled β-L-2′,3′-dd-5′ATP from the monophosphate with near quantitative yields. β-L-2′,3′-dd-5′ATP was a competitive and potent inhibitor of adenylyl cyclases (IC5 ~ 30 nM). Upon uvirradiation β-L-2′,3′-dd-[β-32P]-5′ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2′,5′-dd-3′ATP, a post-transition state, noncompetitive inhibitor. 相似文献
14.
1. The mitochondrial level of AMP gradually diminishes during incubation of mitochondria with glutamate but does not with succinate. This decline of AMP, associated with stoichiometric increase in ADP and/or ATP, is accelerated by the addition of electron acceptors or 2,4-dinitrophenol, while arsenite, arsenate and rotenone are inhibitory. These results are in agreement with the view that AMP is phosphorylated to ADP in the inner space of rat liver mitochondria via succinyl-CoA synthetase (succinate: CoA ligase (GDP), EC 6.2.1.4) and GTP:AMP phosphotransferase dependent on the oxidation of 2-oxoglutarate, which is promoted by the transfer of electron from NADH to the respiratory chain.2. Studies of the periodical changes of chemical quantities of adenine nucleotides as well as of their labelling with 32Pi reveals the following characteristics concerning mitochondrial phosphorylation. (i) In contrast to the mass action ratio of ATP to ADP, the ratio of ADP to AMP is not affected by the intramitochondrial concentration of Pi. (ii) 32Pi, externally added, is incorporated into ADP much more slowly than into γ-phosphate of ATP. (iii) Conversely, ATP loses its radioactivity from γ-phosphate position more rapidly than [32P]ADP when 32P-labelled mitochondria are incubated with non-radioactive Pi.3. In order to elucidate the above characteristic properties of phosphorylation, a hypothetical scheme is proposed which postulates the two separate compartments in the intramitochondrial pool of Pi; one readily communicates with external Pi and is utilized for the phosphorylation of ADP in oxidative phosphorylation, while the other less readily communicates with external Pi and serves as the precursor of ADP via succinyl-CoA synthetase and GTP:AMP phosphotransferase. 相似文献
15.
Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase 总被引:3,自引:3,他引:0
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Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-32P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P2, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-32P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [32P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-32P]ATP or [32P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase. 相似文献
16.
A soluble fraction, obtained by extracting E. coli cytoplasmic membrane vesicles with water, transfers radioactivity from [γ-32P]ATP to a protein present in this soluble fraction. The formation of the [32P]phosphoprotein appears to be reversible. Thus the protein can transfer its 32P to ADP to form [32P]ATP, and the phosphate on the protein can exchange with the phosphate of ATP. Preliminary evidence indicates that the phosphate moiety is linked to a histidine residue of the protein. The Mn2+ and ATP dependencies of [32P]phosphoprotein formation are almost identical to the diglyceride kinase reaction previously reported in intact membrane vesicles. Although indirect evidence supports the involvement of the phosphoprotein in the diglyceride kinase reaction, the soluble fraction catalyzes only a slow formation of [32P]phosphatidie acid from [γ-32P]ATP and α,β-diglyceride. 相似文献
17.
《Bioorganic chemistry》1986,14(2):163-169
The inhibition of Escherichia coli glutamine synthetase by phosphinothricin [2-amino-4-(methylphosphinyl)butanoic acid] has been studied. This amino acid was observed to function as an active site directed inhibitor exhibiting time-dependent inhibition of glutamine synthetase in the presence of ATP or adenylylimidodiphosphate (AMPPNP) but not adenylyl(β,γ-methylene) diphosphonate (AMPPCP). The inactivation was observed to be pseudo-first order. Phosphinothricin was also found to inhibit the enzyme reversibly under initial rate conditions and was competitive with respect to glutamate with K1S = 18 ± 3 μm. The inactive enzyme inhibitor complex was found to contain approximately 11 molecules of ADP and of 32P per dodecamer using [γ-32P]ATP. Reactivation of the inactive enzyme complex was achieved by incubating the enzyme complex in 50 mm acetate (pH 4.4), 1 m KCl, and 0.40 m (NH4)2SO4. ADP, phosphinothricin, and Pi were released upon reactivation. 相似文献
18.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,885(2):195-205
1. The specific activity of the γ-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717–720). The HPLC method also allowed the incorporation of 32P into the (α + β)-positions of ATP to be determined. 2. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [γ-32P]ATP attained a steady-state value after 1–2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the β-agonist isoprenaline increased this value by 5–10% within 15 min. 3. Under these conditions the steady-state specific activity of [γ-32P]ATP was 30–40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the γ-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. 4. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components. 相似文献
19.
A method is described for purification of (N+, K+)-ATPase which yields approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 μmol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600–700 pmol of [3H]ouabain/mg protein when incubated in the presence of either Mg2+ plus Pi or Mg2+ plus ATP plus Na+, and incorporated more than 600 pmol 32P/mg protein when incubated with γ-32P-labeled ATP in the presence of Mg2+ and Na+. The preparation is approximately 35% pure. 相似文献
20.
1. A significant amount of 32Pi is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [32P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of 32Pi into ADP on the one hand and the oxidation of [1-14C]glutamate to 14CO2 on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [32P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [32P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [32P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [32P]ADP. 相似文献