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1.
We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%. 相似文献
2.
A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates. 总被引:1,自引:1,他引:0
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A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor. 相似文献
3.
A method for the synthesis and purification of guanosine 5'-[gamma-S]triphosphate labeled with 32P in the beta-position is described. The first step in the synthesis involves the quantitative transfer of 32Pi from [gamma-32P]dATP to 5'-GMP catalyzed by GMP kinase. Further incubation of the beta-32P]GDP product with [gamma-S]GTP and nucleoside diphosphate kinase results in the synthesis of [beta-32P][gamma-S]GTP with a yield of 10 to 18%. The 32P-labeled [gamma-S]nucleotide is purified by binding to mercury-agarose and eluting with buffer containing beta-mercaptoethanol. Specific incorporation of 32P into the beta-position was demonstrated by treating [beta-32P][gamma-S]GTP with 7% formic acid to remove the gamma-thiophosphate and digesting the remaining [beta-32P]GDP with nucleotide pyro-phosphatase. Although 5'-GMP was released after pyrophosphatase digestion, the only 32P radioactivity detected was as inorganic phosphate. 相似文献
4.
The experiments described in this paper serve as a contribution to the solution of the discrepancies which exist in the assay of ATP:thiamine diphosphate phosphotransferase activity (EC 2.7.4.15), presently in use as a tool for the diagnosis of Leigh's disease (SNE, subacute necrotizing encephalomyelopathy). The results obtained with this phosphotransferase assay can, in part, be explained by the presence of thiamine triphosphate (ThTP) in the preparation of thiamine diphosphate (ThDP) used as a substrate, by the inhibition by ATP of the ThTP phosphohydrolase activity, present in fractions of rat brain homogenates, and by the stimulation by ThDP of the ATPase activity. When [2-14C-thiazole]thiamine was used for the synthesis of [14C]ThTP in fractions of rat brain, it was found that after chromatographic separation of thiamine and its phosphates,14C radio-activity could be demonstrated in the ThTP fractions, even in the absence of an enzyme source. Probably a complex is formed between [14C]thiamine and a phosphate ester which behaves chromatographically as ThTP. It is concluded that the assay system for the measurement of ThTP synthesis in its present form is, in our hands, not suitable for diagnostic purposes. 相似文献
5.
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
6.
We found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed us to synthesize in a single-step procedure carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[gamma-32P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue. 相似文献
7.
8.
Preparation of (beta-32P)ribonucleoside-5'-triposphates using permeable cells of Escherichia coli 总被引:1,自引:0,他引:1
A rapid method for the preparation of [β-32P]ribonucleoside-5′-triphosphates is described. The method involves the incubation of a ribonucleoside triphosphate with 32Pi and E. coli cells made permeable to nucleotides. The labeled triphosphates can be isolated by preparative thin layer chromatography on poly(ethylene)imine cellulose plates. Labeled GTP, CTP, and UTP obtained by this method are more than 99% pure [β-32P]compounds. Labeled ATP contains about equal amounts of label in the β- and γ-phosphate position. Pure [β-32P]ATP can be obtained from this preparation by exchanging the γ-32P against unlabeled Pi and reisolating the labeled ATP by charcoal adsorption and elution. 相似文献
9.
Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents. 相似文献
10.
Tiziana Gigliobianco Bernard Lakaye Alexander F Makarchikov Pierre Wins Lucien Bettendorff 《BMC microbiology》2008,8(1):16
Background
Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. 相似文献11.
Marjorie Gangolf Pierre Wins Marc Thiry Bena?ssa El Moualij Lucien Bettendorff 《The Journal of biological chemistry》2010,285(1):583-594
In animals, thiamine deficiency leads to specific brain lesions, generally
attributed to decreased levels of thiamine diphosphate, an essential cofactor in
brain energy metabolism. However, another far less abundant derivative, thiamine
triphosphate (ThTP), may also have a neuronal function. Here, we show that in
the rat brain, ThTP is essentially present and synthesized in mitochondria. In
mitochondrial preparations from brain (but not liver), ThTP can be produced from
thiamine diphosphate and Pi. This endergonic process is coupled to
the oxidation of succinate or NADH through the respiratory chain but cannot be
energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory
chain inhibitors, such as myxothiazol and inhibitors of the
H+ channel of F0F1-ATPase. It is
also impaired by disruption of the mitochondria or by depolarization of the
inner membrane (by protonophores or valinomycin), indicating that a
proton-motive force (Δp) is required. Collapsing
Δp after ThTP synthesis causes its rapid
disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a
reversible H+-translocating ThTP synthase. The synthesized
ThTP can be released from mitochondria in the presence of external
Pi. However, ThTP probably does not accumulate in the cytoplasm
in vivo, because it is not detected in the cytosolic
fraction obtained from a brain homogenate. Our results show for the first time
that a high energy triphosphate compound other than ATP can be produced by a
chemiosmotic type of mechanism. This might shed a new light on our understanding
of the mechanisms of thiamine deficiency-induced brain lesions. 相似文献
12.
R W McClard 《Analytical biochemistry》1979,96(2):500-503
The straightforward, rapid synthesis and purification of radiochemically pure [1-32P]fructose-1,6-bisphosphate starting from radioactively labeled inorganic phosphate is described. The product has a relatively high specific radioactivity (2 × 105 Ci/mol). Using this method, carrier-free [1-32P]fructose-1,6-bisphosphate could be obtained. The use of [1-32P]fructose-1,6-bisphosphate in the assay of fructose bisphosphatase is illustrated. 相似文献
13.
A new chemical method for the synthesis of adenosine 5'-gamma-[32P] triphosphate has been developed based on the reaction of adenosine 5'-diphosphate with ethyl chloroformate. The resulting active mixed anhydride was able to react with [32P]-triethylammonium orthophosphate to give gamma-[32P]ATP. 相似文献
14.
Abstract: Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 m M ) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P2 fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [β-32 P]TDP and [γ-32 P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn2+ . These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized. 相似文献
15.
In an in vitro incubation, 8-azidoguanosine 5'-[gamma-32P]triphosphate ( [gamma-32P]-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with [gamma-32P]-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, [gamma-32P]-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by [gamma-32P]-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified. 相似文献
16.
Presence of guanine nucleotide-binding proteins in a plant hypocotyl microsomal fraction 总被引:5,自引:0,他引:5
B K Drobak E F Allan J G Comerford K Roberts A P Dawson 《Biochemical and biophysical research communications》1988,150(3):899-903
The presence of specific guanine nucleotide-binding proteins in a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction was investigated. Polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of nitrocellulose blots with [alpha-32P]GTP and [gamma-32P]GTP indicated the presence of four specific and distinct GTP-binding proteins with molecular masses of approx. 23.4 kDa, 24.8 kDa, 26.6 kDa and 28.5 kDa. Binding of [alpha-32P]GTP could be completely prevented by 30 microM GDP or 10 microM guanosine 5'[gamma-thio]triphosphate. This report presents evidence for the presence in a microsomal fraction from zucchini hypocotyls of Gn-proteins as defined by Bhullar and Haslam (1987) Biochem.J. 245, 617-620. The four plant proteins resemble animal Gn-proteins when molecular weights and GTP-binding specificities are considered. 相似文献
17.
43K rapsyn is a peripheral protein specifically associated with the nicotinic acetylcholine receptor (nAChR) present in the postsynaptic membrane of the neuromuscular junction and of the electrocyte, and is essential for its clustering. Here, we demonstrate a novel specific phosphorylation of 43K rapsyn by endogenous protein kinase(s) present in Torpedo electrocyte nAChR-rich membranes and identify thiamine triphosphate (TTP) as the phosphate donor. In the presence of Mg(2+) and [gamma-(32)P]-TTP, 43K rapsyn is specifically phosphorylated with a (32)P-half-maximal incorporation at approximately 5-25 microM TTP. The presence of TTP in the cytosol and of 43K rapsyn at the cytoplasmic face of the postsynaptic membrane, together with TTP-dependent phosphorylation of 43K rapsyn without added exokinases, suggests that TTP-dependent-43K-rapsyn phosphorylation may occur in vivo. In addition, phosphoamino acid and chemical stability analysis suggests that the residues phosphorylated are predominantly histidines. Inhibition of phosphorylation by Zn(2+) suggests a possible control of 43K rapsyn phosphorylation state by its zinc finger domain. Endogenous kinase(s) present in rodent brain membranes can also use [gamma-(32)P]-TTP as a phosphodonor. The use of a phosphodonor (TTP) belonging to the thiamine family but not to the classical (ATP, GTP) purine triphosphate family represents a novel phosphorylation pathway possibly important for synaptic proteins. 相似文献
18.
19.
A simple enzymic method for the synthesis of adenosine 5''-[alpha-32P]triphosphate on a preparative scale. 总被引:10,自引:9,他引:1
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A simple, rapid and inexpensive method is described for the enzymic synthesis of [alpha-32P]ATP from [32P]Pi on a preparative scale with an overall yield of 53%. The final product contained all of the detectable radioactivity (less than 99.9%) in the alpha position and has been shown to behave identically with commerically availabe [alpha-32P]ATP during the synthesis of 3':5'-cyclic AMP in the reaction catalysed by adenylate cyclase. 相似文献
20.
Gangolf M Czerniecki J Radermecker M Detry O Nisolle M Jouan C Martin D Chantraine F Lakaye B Wins P Grisar T Bettendorff L 《PloS one》2010,5(10):e13616