Adenosine tetraphosphate

The enzyme splitting both adenosine-tetraphosphate and guanosine-tetraphosphate was purified to homogeneity from yellow lupin seeds (Guranowski et al., 1997). The polypeptide of 25 kDa catalysed the hydrolysis of nucleoside-5 -tetraphosphate to nucleoside triphosphate and P , and hydrolysed PolyP3, but neither pyrophosphate nor PolyPs The divalent carions Mg2+, Co2+, Ni2+ or Mn2+ were required for the reaction. 

Several novel diadenosine 5, 5" -P-l,P-4-tetraphosphate (AppppA) analogues (94 95) and an adenosine tetraphosphate analogue (%) have been prepared as competitive inhibitors of ADP-induced platelet aggregation. Among the various analogues, the P-2, P-3-monochloromethylene AppCHClppA was found to be particularly active. 

Adenosine tetraphosphate (AP4) (10.112a) and pentaphosphate (Apj) (10.112b) have been identified from several sources, but they do not substitute for ATP (Apj) in phosphorylation reactions and their biochemical role is largely unknown [88,89]. 

Higher polyphosphate derivatives of purine ribonucleosides are known. Adenosine tetraphosphate has been isolated from horse muscle the compound appears to be a 5 -polyphosphate, but the structure of the polyphosphate portion is uncertain and a biological role is unknown (25). Guanosine tetraphosphate and adenosine pentaphosphate have also been reported. 

The adenosyltransferase enzyme of yeast is unable to utilize ADP, adenosine tetraphosphate, or a number of trinucleotides in place of ATP (Mudd and Cantoni, 1958). Recently it has been shown that the a-yS and /3-y methylene analogs of ATP are also inactive, although the former compound inhibits the reaction progressively as the ratio of analog to ATP is increased (Table I) (Mudd, 1970). The structural requirements of the yeast, bacterial, and manunalian enzymes for the amino acid sub-... 

ADP. In the presence of excess phosphoric acid, the ADP product will be phosphorylated to form ATP. Of course, this reaction does not proceed with the same selectivity as the enzyme catalyzed phosphorylation, so that even under optimum reaction conditions, product mixtures of AMP, ADP, ATP, and some higher polyphosphates such as adenosine tetraphosphate are obtained. Nonetheless, the major product is ATP, which may be purified by ion-exchange chromatography ... 

An alternative large-scale synthesis of ATP is one that was recently developed by G. M. Whitesides, M.I.T., which utilizes acetyl phosphate as the phosphate donor and immobilized enzymes (see Section 4.7) as a catalyst (30). The reaction occurs under mild conditions (approximately neutral pH, room temperature), the insoluble polymeric catalyst is easily removed (centrifugation) and need be present only in small amounts, relative to the substrate and the reaction is more specific than a nonenzymatic synthesis (i.e., adenosine tetraphosphate is not formed as a by-product). The net reaction is ... 

Fig. 11. Two-dimensional correlated spectrum of the following mixture ATP, ADP, AMP, inorganic phosphate, pyrophosphate, adenosine tetraphosphate (A4P), diadenosine 5, S"-tetraphosphate (AP4A), and GdCl,. The labeling is consistent with Fig. 10 with the following additions diagonal peaks k and 1 (—23.7 and —23.9 ppm) represent the P- and y-phosphorus resonances of A4P and the p resonances of AP4A, respectively the off- diagonal peak h correlates the

Ap4A, diadenosine tetraphosphate BBG, Brilliant blue green BzATP, 2 - 3 -0-(4-benzoyl-benzoyl)-ATP cAMP, cyclic AMP CCPA, chlorocyclopentyl adenosine CPA, cyclopentyl adenosine CTP, cytosine triphosphate DPCPX, 8-cyclopentyl-1,3-dipnopylxanthine IP3, inosine triphosphate lpsl, diinosine penta phosphate a,p-meATP, a,p-methylene ATP p.y-meATP, p.y-meihylene ATP 2-MeSADP, 2-methylthio ADP 2-MeSAMP, 2-methylthio AMP 2-MeSATP, 2-methylthio ATP NECA, 5 -W-ethylcarboxamido adenosine PPADS, pyridoxal-phosphate-6-azophenyl-2, 4 -disulfonic acid PLC, phospholipase C RB2, reactive blue 2 TNP-ATP, 2, 3 -0-(2,4,6-trinitrophenyl) ATP. 

Selected entries from Methods in Enzymology [vol, page(s)] Adenylate kinase contamination in in phosphotransferases, 63, 7 as contaminant enzyme preparations, 64, 24 P, P -di(adenosine-5 )-tetraphosphate and P, P -di(adenosine-5 )-pentaphosphate inhibition of adenylate kinase, 63, 7, 401, 483. 

In a number of cases there may be a contaminating enzyme present which acts on one or more of the substrates, products, or effectors of the system under study. It may be necessary to include in the reaction mixture a specific inhibitor for that contaminating activity. For example, adenylate kinase is often present in preparations of a number of phosphotransferases. It is often advantageous, in such instances, to include a specific inhibitor of adenylate kinase (e.g., P, P -di(adenosine-5 )-tetraphosphate or P P -di(adenosine-5 )-pentapho-sphate). If an inhibitor of the contaminating activity is added as an additional constituent of the reaction mixture, the investigator should demonstrate that the inhibitor is not an effector of the enzyme under study. 

Quite a different form of exopolyphosphatase was purified from the vacuolar sap of S. cerevisie (Andreeva et al., 1998b). Its molecular mass determined by gel filtration was 245 kDa. This exopolyphosphatase hydrolysed PolyP3 only slightly, and its specific activity increased with the increase in PolyP chain length (Table 6.6). It was unable to hydrolyse adenosine- and guanosine-tetraphosphates and was insensitive to antibodies inhibiting the low-molecular-mass exopolyPase of the cytosol (Table 6.4). This enzyme was stimulated by divalent metal cations to a much lesser extent than 40 kDa exopolyphosphatase (Table 6.5) and was inhibited by EDTA (Table 6.4). The inhibitory effect of EDTA is explained by the binding of Co2+, which is the best activator of the vacuolar exopolyphosphatase at 0.1 mM. 

The enzyme with adenosine-tetraphoshatase activity was obtained earlier from rabbit muscle (Small and Cooper, 1966). This enzyme had an effect on inosine tetraphosphate and tripolyphosphate but showed little or no activity with other nucleotides or PolyPs. 

A. Guranowski, E. Starzynska, P. Brown and G. M. Blackburn (1997). Adenosine 5 -tetraphosphate phosphohydrolase from yellow lupin seeds purification to homogeneity and some properties. Biochem. J., 328, 257-262. 

T. V. Kulakovskaya, N. A. Andreeva and I. S. Kulaev (1997). Adenosine-5 -tetraphosphate and guanosine-5 -tetraphosphate - new substrates of the cytosol exopolyphosphatase of Saccharomyces cerevisiae. Biochemistry (Moscow), 62, 1180-1184. 

With these points in mind, some characteristics of the receptors are noted, particularly their sensitivities to the natural nucleotides ADP (adenosine diphosphate), ATP (adenosine triphosphate), UTP (uridine triphosphate), UDP (uridine diphosphate) and Ap4A (Ap(4)A P1.P5-diadenosine tetraphosphate). 

Several inhibitors are known that bind to the adenosine site of PARP (19). These inhibitors include 6-bromo-2 -deoxyuridine, caffeine, 5-bromouracil, diadenosine-tetraphosphate, 1-methyladenine, 5-nitrouracil, theophylline, theobromine, thymidine, and other compounds. These compounds are not as well studied as the nicotinamide analogs. In addition, it is not known whether these compounds can interact with the adenosine (A,) receptors that are involved in modulation of synaptic transmission and neuroprotective effects. 

The recent establishment of a link between ribonucleotide metabolism, specifically the synthesis of the cellular metabolite, diadenosine tetraphosphate, and its relationships with adenosine diphosphate-ribosylation reactions in the cell, presents a significant new target. 

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