Venom 5 -nucleotidase

Ribonucleoside 5 -0-hydroxymethylphosphonates (8 R = OH) are resistant to the action of phosphatases and phosphodiesterases. They are, however, good substrates for snake venom 5 -nucleotidase, unlike (8 R = H).2 ... 

The assay is carried out both at 1 jaM and 1 mM substrate concentrations [162]. The enzyme preparation is incubated with tritium-labelled cyclic nucleotide in the presence of Mg, the reaction is stopped by brief heating in a water bath, the 5 -nucleotide formed is converted to the nucleoside by snake venom 5 -nucleotidase, the nucleoside is separated from the remaining cyclic nucleotide by anion exchange chromatography (Dowex 2), and the radioactivity of both compounds is counted. 

The entire assay can conveniently be carried out in a glass scintillation vial [82]. The reaction volume contains Tris buffer (pH 7.5), Mg, [ H]cyclic nucleotide, and snake venom 5 -nucleotidase. After a 10-min incubation at 37°C, the reaction is stopped by the addition of a slurry of AG1-X2 anion exchange resin. The resin binds substrate cyclic nucleotides but does not bind nucleosides. Scintillation fluid is added and the amount... 

Condensation of A -benzyloxycarbonylaminomethanephosphoric acid with 2, 3 -0-isopropylidene nucleosides in the presence of an aryl sulphonyl chloride gives the corresponding phosphonates, which on treatment with aqueous hydrobromic acid lead to the nucleotide analogues (10). Although (10) are resistant to alkaline phosphatase, they are degraded slowly by snake venom 5 -nucleotidase. Uridine 5 -phosphofiuoridate is also degraded slowly by the latter enzyme whereas it is resistant to the former. ... 

Treatment of bromomethyl acetate with the sodium salt of a dialkyl phosphite, followed by deacetylation with methoxide affords the corresponding dialkyl hydroxymethanephosphonate. If this is tosylated with tosyl chloride, and the product treated with nucleosides [protected at the 3 -(and 2 -, if present) hydroxy groups] and sodium hydride in DMF, the monoalkyl ester of a 5 -0-phosphonyl-methyl nucleoside (60) is obtained after deblocking the sugar, and subsequent dealkylation with TMS-iodide affords (61). Like alkyl esters of 5 -mononucleotides, (60) is resistant to acid and alkaline hydrolysis, while (61) is stable in acidic and alkaline media, and is also resistant to hydrolysis by alkaline phosphomono-esterase and snake venom 5 -nucleotidase. Treatment of (61) with DCC and morpholine, and subsequently with orthophosphate or pyrophosphate, affords (62) and (63), respectively. Alkaline phosphomonoesterase from E. co/i hydrolyses the pyrophosphate links in (62) and (63) to give (61) and orthophosphate. The UTP and CTP analogues (63 B = U or C) are inhibitors of uridine kinase from... 

Dhananjaya BL, Nataraju A, Gowda CDR, Sharath BK, D Souza CJM (2009) Vanillic acid as a novel specific inhibitor of snake venom 5 -nucleotidase a pharmacological tool in evaluating the role of the enzyme in snake envenomation. Biochemistry 74 1315... 

By using highly specific radioactive cyclic nucleotides and the snake venom nucleotidase, the most sensitive, specific and convenient method of assay for phosphodiesterase has been evolved. 

As for the stingray venom, not much is known. There was a report on the presence of 5 -nucleotidase and phosphodiesterase in stingray, Urolophus halleri, venom (29). 

The bearing which these discoveries have had on the elucidation of the structure of ribopolynucleotides will be discussed later. It is important to stress here, however, that, for most purposes, the older methods of preparing nucleotides have been superseded by procedures which yield separate isomers of each. Of the techniques mentioned above, paper chromatography iB mainly of analytical value, and is the most convenient method for the qualitative detection of isomeric adenylic acids. The only disadvantage of this method is that the isomers are not completely separable from muscle adenylic acid. The presence of the latter, however, can be readily detected by hydrolyzing it to adenosine by means of the specific 5-nucleotidase present in snake venoms,66 or by deamination by a specific enzyme... 

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

First, exonuclease (phosphodiesterase), endonuclease (8, 10-12), 5 -nucleotidase (13), and nonspecific phosphatase (13) are present in all venoms that have been analyzed. Therefore, nature did not provide these materials as sources of any one of these enzymes free from undesirable contaminants which may be represented by the others. 

Several methods for preparation of purified venom exonuclease have been described [see the reviews on methods (15, 18, 19, 21) and books (22-24)]. The major effort of purification was directed toward removing the contaminating monophosphatases. A successful and widely used step was introduced by Sinsheimer and Koerner (43). At pH 4, monophosphatases are precipitated with a lower concentration of acetone the remaining exonuclease is precipitated by a higher acetone concentration. Several modifications of this principle have been proposed (concentration of acetone, temperature during precipitation, etc.). Commercially available preparations represent essentially this stage and contain per unit of exonuclease 10 4 unit of 5 -nucleotidase, 10 3 unit of nonspecific phosphatase, and about the same amount of endonuclease. The last figure is only an approximation because of the difficulty of accurate determination. The more elaborate preparations have these contaminants... 

It has been known for many years (15-24) that venom exonuclease is capable of hydrolyzing both DNA and RNA. Gray and Lane (56) showed that naturally occurring 2 -0-methyl-substituted ribose derivatives are also hydrolyzed, even though the rate of hydrolysis is slower than with the unsubstituted ribose. Interestingly, the 2 -0-methylated derivatives are totally resistant to 5 -nucleotidase (57, 58). 

Nucleotidase (5 -ribonucleotide phosphohydrolase, EC 3.1.3.5) is widely distributed in nature and a voluminous literature has appeared in the past decade on the enzyme from vertebrate tissues, seminal fluid, snake venoms, yeasts, and bacteria. Studies regarding the discovery and early investigations of the enzyme have been reviewed by Heppel (1) and... 

Figure 9.93 HPLC chromatograms of phosphomonoesterase hydrolysis of A(S)MP. (i4) Chromatogram obtained from calf intestinal mucosa alkaline phosphatase hydrolysis of A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-HCl (pH 8.1), 300 pM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 2 of enzyme and incubated at 30°C for 6 hours. A 20 /xL sample was then injected onto the HPLC column and analyzed. (B) Chromatogram obtained from snake venom S -nucleotidase incubated with A(S)MP. In a reaction volume of 100 /xL containing 100 mM Tris-Cl (pH 8.1), 300 jxM A(S)MP, and 20 mM MgCl2, the reaction was initiated by addition of 6 yxg of enzyme and the reaction mixture incubated at 30°C for 60 minutes, and a 20 yxL sample was injected onto the HPLC column and analyzed. (From Rossomando et al., 1983.)...

The racemic ( )-3TC 232 and other pyrimidine and pnrine analogues were resolved by varions methods (such as separation on HPLC with a chiral column, enzyme-catalyzed hydrolysis of its 5 -0-butanoyl ester derivative. or enzy matic resolution of its monophosphate derivative with a nucleotidase derived from Crotalus atrox venom ). Resolution of racemic thia sugar precursors lias also been described. 

Enzymatic degradation is often useful. Pyrophosphatases have been used to produce the sugar-1-phosphate and the nucleotide. Treatment of the former with a phosphatase will give the free sugar, while the nucleotide can be broken down to the nucleoside with 5 -nucleotidase. Snake venoms have been much used, since they can contain both pyrophosphatase and 5 -nucleotidase. 

In addition to the relatively unspecific P, some P show a high specificity, e.g. 5 -nucleotidase from snake venom and 3 -nucleotidase from rye, which catalyse the hydrolysis of 5 -nucleotides or 3 -nucleotides to their respective nucleosides (see Purine degradation, Pyrimidine degradation). 

Two enzymes were selected In which proper alignment of nucleotide C3 and base moieties Is necessary for their catalytic action AMP amlnohydrolase (AMP deaminase, EC 3>3.4.6) from rabbit muscle requires substrates with an anion at C5 and catalyses deamination at C6-NH2 of adenine [42]. Vice versa, In snake venom 3 -nucleotidase (EC 3>1 3.5 hydrolysis occurs at the 5 -phosphate vdilch has to be at a defined distance from the base, as Judged by the Inactivity of 3 -phosphates [43]. 

Structure of Coemyme A. The elucidation of the structure of CoA depended heavily on d radation by specific enzymes. The phosphate on carbon 3 of the adenosine was shown to be a monoester phosphate by hydrolysis with prostate phosphomonoesterase. The localization of the monoester at the 3 position was established by its sensitivity to a b nucleotidase that attacks only nucleoside 3 -pbosphates, not 2 - or 5 -phosphates. The original CoA molecule or the phosphatase product, depbospho CoA, can be split by nucleotide pyrophosphatases from potato or snake venom. These reactions permitted the identification of the adenosine phosphate portion of the molecule. The position of the phosphate on pantothenic acid cannot be determined enzymatically, but was established by studies on the synthesis of CoA from synthetic phos-phorylated pantetheines. Pantetheine is split to thiolethanolamine and pantothenic acid by an enzyme found in liver and kidney. This enzyme also attacks larger molecules, including CoA. 

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