DNases venom phosphodiesterase

Venom exonuclease [EC 3.1.15.1], also known as venom phosphodiesterase, catalyzes the exonucleolytic cleavage of RNA or DNA (preferring single-stranded substrates) in the 3 to 5 direction to yield 5 -phosphomononucleo-tides. Similar enzymes include hog kidney phosphodiesterase and the Lactobacillus exonuclease. See also specific phosphodiesterase J. A. Gerit (1992) The Enzymes, 3rd ed., 20, 95. 

Snake Venom Phosphodiesterase An exonuclease is an enzyme that sequentially cleaves nucleotides from the end of a polynucleotide strand. Snake venom phosphodiesterase, which hydrolyzes nucleotides from the 3 end of any oligonucleotide with a free 3 -hydroxyl group, cleaves between the 3 hydroxyl of the ribose or deoxyribose and the phosphoryl group of the next nucleotide. It acts on single-stranded DNA or RNA and has no base specificity. This enzyme was used in sequence... 

The reaction of 5 -amino-5 -deoxyadenosine with trimetaphosphate affords the 5 -Af-triphosphate (23). When (23) is employed as substrate with glucose in the hexokinase-catalysed reaction, the 5 -AT-diphosphate (24) is obtained the latter is cleaved by snake venom phosphodiesterase to the 5 -phosphoramidate, and hydrolyses in acid to the amino-nucleoside. It does not appear to be polymerized by polynucleotide phosphorylase. In this context it is noteworthy that uridine 5 -5-thiopyrophosphate (25) is a competitive inhibitor for polynucleotide phosphorylase from E. coli, but not a substrate, and that the 5 -S-thiotriphosphates (26) and (27) show neither substrate nor inhibitory properties for RNA polymerase or DNA polymerase I, respectively. However, (23) can be polymerized using the latter enzyme, showing that the introduction of a 5 -heteroatom does not completely exclude these modified nucleotides as substrates for the polymerizing enzymes. 

ADP-ribose) synthetase and the latter fraction involves ADP-ribosyltransferase. To clarify that the radioactive compound formed by the latter fraction was indeed the mono(ADP-ribose) molecule, the acid-insoluble reaction product was treated with alkali at 37°C for 2 h. The radioactive material solubilized was adjusted to pH 7.0 and subjected to high performance liquid chromatography with reverse phase column. The eluate was monitored by UV and fractionated and radioactivity of the fraction was measured. The retention time of the radioactive product coincided with that of authentic mono(ADP-ribose). Furthermore, by treatment with snake venom phosphodiesterase this radioactive peak, tentatively considered to be ADP-ribose, migrated to the position corresponding to the 5 -AMP. These results indicate that hen liver nuclei contain ADP-ribosyltransferase. We purified this enzyme to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose [3]. 

The mechanism of inhibition by intercalators in the absence of DNA-histone complexes is not yet clear. However, the inhibition by ethacridine is probably due to its binding to the substrate, poly(ADP-ribose). This inference is drawn from the two observations that ethacridine inhibits the degradation of poly(ADP-ribose) not only by the glycohydrolase but also by the snake venom phosphodiesterase, and that ethacridine prevents the ethanol-acetate precipitation of poly(ADP-ribose). In the presence of DNA-histone complexes all inhibitory dyes produce a similar inhibition of the enzyme activity at equimolar concentrations. This suggests that the inhibition in these cases is due to the presence of the same amount of histones released by different intercalators. 

Poly(ADP-ribose) was purified almost to homogeneity fi om bull testes by the method of Agemori et al. (5). Purified polymerase was incubated briefly with [ P]NAD (10°C, 30 sec, 5 xM NAD) in the presence of 10 pg/ml Hae IH-digested calf thymus DNA. Incorporation was stopped by the addition of 3-aminobenzamide and unincorporated labeled NAD was removed. Pulse-labeled polymer was originally attached to the enzyme. After alkali treatment it showed a heterogeneous distribution of short chains on a 20% polyacrylamide gel (6). Venom phosphodiesterase digested these to p2p]pR AMP and [ P]AMP, of which AMP represented 4.6% of the counts. After a chase with unlabelled NAD (200 pM NAD, 10 min, 25 °C),... 

Phosphoramidates. Phosphoramidates are another commercially available DNA analogue where the 3 -oxygen of the phosphodiester center is replaced with an amino or aminoalkyl group (Fig. 13) (49). Phosphoramidates exhibit complete resistance to nuclease PI and snake venom phosphodiesterase and a moderate improvement in affinity for DNA and RNA targets (ATm per monomer = 4°C for 2 -fluoro N3 -P5 phosphoramidates) (57). However, they exhibit no RNase H activity (49). 

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... 

Spleen (and micrococcal) phosphodiesterase. These exonucleases act on DNA and RNA in a direction that is the reverse of snake venom phosphodiesterase, i.e., they start from the 5 -OH and yield sequentially 3 -phosphates. They... 

Stable hybrids with DNA and may enter cells through passive transport mechanisms. Finally, and potentially important for drug delivery, we have shown that the boranophosphates are nuclease resistant (12), like the phosphorothioates and methyIphosphonates. Short boranophosphate oligomers exhibit a high degree of stability to spleen phosphodiesterase and snake venom phosphodiesterase, indicating that they could have long half-lives in the cell. 

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