Snake venom phosphatases

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 ... 

SVPDE/AP snake venom phosphodiesterase and alkaline phosphatase t-BOC tm-butoxycarbonyl (group)... 

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.)...

Hall (1964) used whole snake venom and bacterial alkaline phosphatase to prepare nucleosides from mixed sRNA in a search for minor bases (Hall 1965). 

That the phosphorus is pi esent as a monoester is further supported by the finding that on pretreatment of ovalbumin with the phosphodiesterase from snake venom and subsequent incubation at pH 5.6 with prostate phosphatase, the same amount of phosphorus is released as with this enzyme alone, i.e., 46%. Moreover, no change in the electrophoretic behavior occurs (72). 

If a-casein is treated with either the crystalline pyrophosphatase of yeast at pH 7.0 (35) or with the snake venom diesterase at pH 8.2 (87), no inorganic phosphorus is released. However, if the diesterase reaction is carried out in weakly buffered solutions a small drop of pH takes place (71), indicating the exposure of acidic groups. Subsequent incubation of the diesterase-treated a-casein with prostate phosphatase at pH 6.0 hberates no more phosphorus than in the absence of the diesterase. If, how ever, prostate and intestinal phosphatase are added, 78 % of the a-casein phosphorus is set free. Since the intestinal enzyme at pH 6.0 acts on low... 

Action of Prostate Phosphatase on -Casein Pretreated with Snake Venom Diesterase... 

As phosphodiesters, both RNA and DNA are subject to hydrolysis by nonspecific phosphodiesterases. Historically, the enzymes of greatest interest in this area are snake venom phosphatases. Certain venoms con-... 

The syntheses of dithymidylyl-3, 5 -phosphorofluoridate and phosphorothiof-luoridate (87) and (88) have been described. These involved the fluorinolysis of the P-Se bond in the bis-dimethoxytrityl selenomethyl esters (89) and (90). Compounds (87) and (88) were reported to be hydrolytically unstable, with no inhibitory activity on the snake venom and spleen phosphodiesterases and alkaline phosphatases. Finally, neither was considered as a highly toxic dinuc-leotide. ... 

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. ... 

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 a second procedure, poly(ADP-ribose) was first separated from the bulk of the nucleic acids and proteins by dihydroxyboryl-Sepharose affinity chromatography 147,190). The isolated polymer was treated with snake venom phosphodiesterase and bacterial alkaline phosphatase to yield the nucleoside 2, l"-ribosyladenosine from internal residues. This product was then treated with chloroacetaldehyde to produce the fluorescent derivative, l,iSr -ethenoribosyladenosine, which was then separated from other derivatized residues by reversed-phase high performance liquid chromatography picomole amounts were quantified by fluorescence detection. This procedure facilitates the accurate determination of minute quantities of endogenous poly(ADP-ribose) (102, 190). Niedergang et al. (147) have also utilized a fluorimetric assay for determination of the enzymatic digestion products of the polymer, ADP-ribose, or iso-ADP-ribose. 

Digestion with snake venom phosphodiesterase, wheat germ acid phosphatase, ans beef spleen phosphodiesterase are almost extended to 77 83%, 97 99%, and 77 80%, respectively. Further no evidence was found for the presence of oligonucleotides linked through the heterocyclic base. 

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. 

Furthermore, incubation of APh-GDP with alkaline phosphatase (intestinal) and phosphodiesterase (snake venom) liberates 4-azidophenol. This leads to an increase in absorption at 303 nm. Incubation with phosphodiesterase alone produces APh-P, and incubation with alkaline phosphatase alone is without effect. 

The 5 -Substituted Nucleotides. These are adenosine-5 -phosphate, guanosine-5 -phosphate, uridine-5 -phosphate, and cytidine-5 -phosphate. All of them have been found among the hydrolysis products when ribonucleic acid is treated with snake venom diesterase or with intestinal phosphatase in the presence of arsenate. These compounds contain phosphate esterified at carbon 5 of the ribose moiety. 

Desoxyribonucleic acid is hydrolyzed by calf intestinal diesterase to give a mixture of mononucleotides.Phosphatase activity present in the enzyme preparation must be inhibited by arsenate otherwise, nucleosides are obtained. The same nucleotide mixture results from the action of snake venom diesterase freed of phosphatase activity by chromatography on a cellulose column." ... 

Intestinal mucosa enzyme partially inhibited by arsenate leads to the formation of guanosine, cytidine, and cytidine-5 -phosphate. This preparation splits at (2) with subsequent partial dephosphorylation. Snake venom diesterase also sphts at (2). Prostatic phosphatase liberates... 

The digestive origin of the secretion is further borne out by the presence in snake venoms of such hydrolases as proteases, peptidases, phosphatases, sterases, and lecithinases. The new specialization expresses itself by the presence of hyaluronidase, assuring the diffusion of the venom, and by the presence of substances of high toxicity (see Zeller, 1948). 

Snake venom phosphodiesterase (SVPDE)/alkaline phosphatase (AP). 0.3 A260 U oligonbonucleotide 50 pL of 50 mM Tris-HCl/10 mM MgClz (pH 8.0)... 

Nonspecific Phosphoesteroses. These enzymes cleave a variety of phosphoric acid derivatives, even synthetic substrates and lecithins (diesters of phosphoric acid with glycerol and choline). Such enzymes occur among other places in snake venom, and in the intestinal mucosa. Snake venom phosphatases specifically split 3 -phos-phate bonds, giving rise to 5 -monophosphates, whereas chemical hydrolysis yields a mixture of the 2 - and 3 -monophosphates. Free 3 -phosphate groups (the monoesters) are inhibitory in the enzymic reaction. 

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