Snake venom diesterase

Digestion of this substance XIX with a snake-venom diesterase which also contained a strong pyrophosphatase gave thymidine 5-phosphate (XX)... 

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... 

As is shown in Table VI, most of the phosphorus of /3-casein is liberated by the prostate enzyme at pH 6.0 after pretreatment with the snake venom diesterase. This indicates that the /3-protein contains diester bonds of the —0—P—0— type, which is in contrast to the —N—P—0— of the a-casein (71). 

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

The first RNA molecule to be sequenced was not a virus, but a tRNA. Two enzymes were required in these analyses (i) bovine pancreatic ribonuclease, which cleaved after pyrimidines (C or U), became a classic system for scientific studies after Armour Co., the hotdog Company, purified 1 kg of the enzyme and distributed it to scientists (source Wikipedia) and (ii) takadiastase ribonuclease Tl, which cleaved 3 to a guanosine (G). Each of these small fragments was further analyzed by partial digestion with snake venom diesterase from the 3 -ends. Once again, biochemistry and enzymology led to breakthroughs in chemical analysis. 

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. 

Pyrimidine Nvdeoside Diphosphates. When ribonucleic acid is digested with purified snake venom diesterase (free of phosphomono-esterase activity) approximately 20% of the total base can be accounted for as 2, 5 and/or 3, 5 diphosphates of cytidine and uridine. The mechanism of origin of these compounds is discussed below. 

Snake venom diesterase and intestinal mucosa diesterase are thought to cleave the phospho-diester bond, as indicated by the dotted lines in (VIII). It can be seen that splitting in this manner would yield a mixture of nucleoside-5 -phosphates. 

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 essentiality of an intact adenylyl end group for amino acid acceptor activity of soluble RNA from E. coU was also demonstrated by Preiss and co-workers (194) iu a number of ways. First, they showed that periodate, which splits cis-hydroxyls and hence, in a polyribonucleotide, can act only on the terminal nucleotide containing a free 3 -OH, completely inactivated their RNA. Second, snake venom diesterase, which degrades a polyribonucleotide chain starting from the end bearing the 3 -hydroxyl group caused complete loss of amino acid acceptor activity en approximately... 

Snake venom phosphodiesterase and polynucleotide phos-phorylase. Snake venom diesterase is an exonuclease that hydrolyzes both DNA and RNA it can be isolated from the venom of many poisonous snakes. It attacks sequentially from the 3 -OH end and yields 5 -phosphates. Polynucleotide phosphorylase (PNPase) has a similar mode of action. As the name indicates, it is a phosphorylase and not a... 

In addition to the proposed regulatory role of ATP and pyrophosphate, some possibility exists that 3, 5 -cyclic phosphate diesterase is under physiological control. Such ideas arose through observations of Cheung (43, 62) that the partially purified enzyme from beef brain was markedly activated by snake venom. The stimulatory factor was labile at extreme pH it was not dialyzable and appeared to be a protein. A similar activating factor is also present in brain tissue (63) and is removed during purification of the diesterase. It seems to interact stoichiometrically with the enzyme. The activator is destroyed by trypsin and is not proteolytic itself. The precise role of this protein in regulating the phosphodiesterase in vivo is not yet established, however. 

The inactivated 3, 4 -dideoxykanamycin B in the reaction mixture was extracted by two repetitions of chromatography on Amberlite CG-50 (NHa ) resin, with 0.2% ammonia for elution, and the product purified by chromatography on CM-Sephadex C-25 with ammonium formate. The inactivated product was eluted with 0.8 M ammonium formate, and separated from the salt by chromatography on Amberlite CC-50 resin with 0.2% ammonia. The inactivated product thus purified darkens at 205-209°, but does not melt even at 280°. The empirical formula of monoadenylyl-3, 4 -dideoxykanamycin B trihydrate was shown by elementary analysis. It gave positive ninhydrin, Rydon-Smith, and Hanes " reactions. On high-voltage paper-electrophoresis at 3.5 kV for 15 minutes, with 3 1 36 acetic acid-formic acid-water, the inactivated product moved 13.4 cm towards the cathode, whereas 3, 4 -dideoxykana-mycin B moved 16.8 cm. The inactivated product showed a u.v. maximum at 260 nm (c iv 15.40) in water, and a maximum at 258 nm 14.40) in 0.1 M hydrochloric acid. The inactivated product was hydrolyzed to 3, 4 -dideoxykanamydn B and adenylic acid by snake-venom phosphate diesterase. 

Venom Phosphodiesterase. A phosphodiesterase from the venom of several species of snakes exhibits extreme specificity with regard to nucleotides. It hydrolyzes only components from a phosphate esterified at a 5 position. Thus, 5 nucleotides are liberated from RNA. The venom diesterase attacks both purine and pyrimidine nucleotides (Fig. 26). This activity was useful in establishing that 3 -5 linkages, rather than 2 -3 linkages, occur in nucleic acids. A similar enzyme occurs in intestines. 

That being so, some of the common nucleases which degrade ordinary polynucleotides could well have evolved to interact primarily with polynucleotides whose nucleoside components are anti. It is of interest, in this regard, that the diesterases from spleen and snake venom, and micrococcal nuclease, do not digest poly F, which is syn also that the specificity of pancreatic RNase for the natural substrates - uridine and cytidine - is based on their normal anti conformation. 

With the fruitful collaboration of Leon Heppel, a polynucleotide was prepared from the four NDPs including a- P-labeled ADP (Fig. 2). Hydrolysis by snake venom phosphodiesterase brealcs the C—P bond indicated by the arrows in Fig. 2. The labeled products obtained from the synthetic polynucleotide were exclusively P-AMP, indicating the same 3 -5 phosphodiester bond as in natural RNA. Spleen diesterase, which cleaves the phos-phodiester bonds as shown in Fig. 3, produced P-labeled UMP, CMP, GMP and AMP. Thus there was a transfer of label ( nearest-neighbor ) as would be found in RNA. 

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