Phosphodiesterases spleen

Chlamydomonas nuclease. Spleen phosphodiesterase Spleen endonuclease] (3.1.31.1) is produced and catalyzes the reaction Endonucleolytic cleavage to 3 -phosphomono- and oligonucleotide end products. [Hydrolyses double- or single-stranded substrate. Formerly EC 3.1.4.7.]... 

Viral RNA, RF-RNA and 1 % were exposed to RI-RNA formaldehyde (Bishop and Koch, 1967, 1969), to UV (Koch, Quintrell and Bishop, 1967), to heat (80° C) in phosphate buffered saline (Koch, unpublished), to 0.5% diethylpyrocarbonate at 0° C (Breindl and Koch, I972) or to RNase A and exonucleases (RNase II, venom phosphodiesterase, spleen phosphodiesterase) (Mittelstaedt and Koch, 1974). 

FIGURE 11.31 Snake venom phosphodiesterase and spleen phosphodiesterase are exonncleases that degrade polynncleotides from opposite ends. 

Phosphoramidate analogues of dideoxyribonucleoside phosphates (26) and trideoxyribonucleoside phosphates are acid labile and can be hydrolysed enzymically. Snake venom phosphodiesterase cleaves (26) to thymidine and 5 -deoxy-5 -aminothymidine (27 R = H). The latter presumably arises by spontaneous decomposition of the phosphoramidate (27 R = PO3H2) and P—O fission must have occurred during the initial hydrolysis. With acid or spleen phosphodiesterase, (26) gave Tp and (27 R = H), i.e. P—N fission occurred. 

The question of enzyme specificity for irradiated polynucleotides is taken up in more detail in the recent review of Johns.11 The specificities of four enzymes, spleen phosphodiesterase, snake venom phosphodiesterase, pancreatic ribonuclease, and pancreatic deoxyribonuclease are discussed. 

Bovine spleen phosphodiesterase hydrolyzes both polyribo- and polydeoxyribonucleotides... 

Apart from important similarities in the endo- and exonucleolytic properties of staphylococcal nuclease and other well-studied phosphodiesterases (67), those from snake venom and spleen, the basic structural substrate elements for these enzymes appear to be quite different... 

Fig. 2. Proposed structural requirements for substrates of phosphodiesterases that hydrolyze DNA and RNA, those from (a) snake venom, (b) spleen, and (c) staphylococcus (R = thymine and R — p-nitrophenyl). The studies indicated for the venom and spleen enzymes are those suggested by Khorana (67) [data from Cuatrecasas el al. (61)).

The enzyme has also been called spleen phosphodiesterase (1,2) and phosphodiesterase II (3). We prefer to use the term phosphodiesterase as a general name for the broad group of enzymes hydrolyzing phos-phodiester bonds whether between nucleosides or not (4). Table I gives a few examples of such enzymes. The term phosphodiesterase II (3), intended to mean an enzyme releasing nucleoside-3 -phosphates, seems to be an unhappy one, like that of deoxyribonuclease II (5) from... 

Other substrates for spleen exonuclease are the p-nitrophenyl esters of nucleoside-3 -phosphates and bis(p-nitrophenyl) phosphate, which is split only very slowly. These substrates are also split by enzymes having quite different natural substrates (Table I) (80-87). In fact, not only phosphodiesterases, in a broad sense, such as acid DNase, micrococcal nuclease, spleen and venom exonucleases, and cyclic phosphodiesterase but also enzymes such as nucleoside phosphoacyl hydrolase and nucleoside polyphosphatase split these substrates. As pointed out by Spahr and Gesteland (86), this may be explained by the fact that these substrates are not true diesters but rather mixed phosphoanhydrides because of the acidic character of the phenolic OH. It is evident that the use of the synthetic substrates, advocated by Razzell (3) as specific substrates for exonucleases, may be very misleading. Table II shows the distinctive characters of three spleen enzymes active on bis(p-nitrophenyl) phosphate which are present in the crude extracts from which acid exonuclease is prepared. 

As mentioned briefly above, the enzymatic excision of damaged nucleobases may cause some problems. A case in point is the action of nuclease PI. While a single 8-oxo-G lesion is excised as the damaged nucleoside, the clustered 8-oxo-G/Fo lesion is only obtained as modified dinucleotide (Maccubbin et al. 1992). Another example is the hydrolysis of dG pC which severely inhibits the action of bovine spleen phosphodiesterase, while HMUrapA shows only very little inhibition (Maccubbin et al. 1991). Enzymatic hydrolysis of DNA is, in fact, the recommended method for the determination of HMUra (Teebor et al. 1984 Frenkel et al. 1985). It is recalled that mammalian cells cope with this DNA lesion with the help of a hydroxymethyluracil glycosylase (Hollstein et al. 1984). 

Since it is well established that the P-chiral phosphorothio-ates serve as effective probes in mechanistic studies for the phos-phoryl group transfer enzymes /16/, we turned our attention to the application of diastereomeric 8 (B=Thy, Ar=pN02C6Hi -,/17f) to elucidate the mode of action of spleen phosphodiesterase (SPDE, EC 3.1.1. 18). This enzyme splits the phosphodiester bonds to yield nucleoside 3 -phosphates. In the case of it was expected that its SPDE-catalyzed hydrolysis in 180 H 2O medium leads to P-chiral thymidine 3 - 180 phosphorothioate. On the contrary to our expectation the main product of this reaction was thymidine cyclic 3 ,5 - Rp phosphorothioate (10) /6/. By treatment of 8 under the same conditions, but in the absence of the enzyme, no trace of J 0 was detected. ... 

All three of the fully-unblocked dodecamers underwent complete digestion to give their monomeric components when they were treated with Crotalus adamanteus snake venom and spleen phosphodiesterases. Their structures were further confirmed in the usual way. 

Fig. 2.8. Nearest neighbour analysis and quantitative depurination analysis of a defined product from a primed synthesis reaction. When radioactive dATP (or dGTP) is used in the primed synthesis, depurination analysis will yield pyrimidine tracts each of which terminate in a radioactive 3 -phosphate. Thus only those depurination products which lie 5 -adjacent to the labelled nucleotide will be labelled. Each depurination product will be labelled to the same specific activity thus greatly simplifying the quantitation. Digestion of the labelled product with a mixture of micrococcal nuclease and bovine spleen phosphodiesterase yields the nucleoside 3 -monophosphates. Identification of the labelled products (by paper electrophoresis at pH 3.S) gives the nearest neighbours to the labelled substrate.

Fig. 2.11. In (a), 3 -end labelling is achieved using deoxynucleotidyl transferase and an ff-[32P] ribonucleotide triphosphate followed by elimination of the ribonucleotide residues. In (b), 5 -end labelling is carried out using polynucleotide kinase and y-[32P]ATP. Partial digestion with spleen phosphodiesterase removes nucleotides sequentially from the 5 -end of the oligonucleotide giving a mixed population of partially degraded molecules each labelled at the 3 -end. Venom phosphodiesterase removes nucleotides from the 3 -end similarly yielding a mixed population of shortened fragments. The products of the reaction are resolved by two-dimensional electrophoresis-homochromatography and the sequence deduced by the characteristic...

Phosphodiesterase II Bovine spleen RNA and DNA (single-stranded) exonudease splits d linkages at 5 end, releasing 3 -phosphonudeosides... 

Tritylagarose (29) binds enzymes in a reversible fashion through hydrophobic forces, providing another kind of reversible immobilization. Several enzymes, such as alkaline phosphatase, fl-galactosidase, lactate dehydrogenase, and spleen phosphodiesterase, have been immobilized (29). 

Two diastereoisomers have been identified as radiation induced decomposition products of dC. The synthesis of one of these isomers, (5 5,55,6S)-5, 6-cyclo-5-hydroxy-5,6-dihydro-2 -deoxyuridine (81), and its incorporation into DNA, has been reported The presence of the lesion causes a marked destabilisation of DNA duplexes. The nucleases PI, calf spleen phosphodiesterase and bovine intestinal mucosa phosphodiesterase failed to cleave the lesion, nor was it a substrate for a number of repair enzymes, and it acted as a block to Klenow fragment and Taq polymerases. 

This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease II. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows p-nitrophenyl phosphate, 100 5 -AMP, 63 j8-glycerophosphate, 60 ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 and pH 4.8. The Michaelis constant at 37°C was 7.25 X 10" mM. Phosphate and chloride ions acted as competitive inhibitors. 

Bovine spleen phosphodiesterase hydrolyzes both polyribo- and polydeoxyribonucleotides Figure 5-43 Some hydrolytic cleavage reactions of polynucleotides. Reactions of both RNA and DNA are included. 

Furfuryl-8-azapurine was found to be 100 times as potent as theophylline in inhibiting the cyclic phosphodiesterase of lipocytes and in potentiating the stimulation of lipolysis. 8-Azaguanine (25 mg/kg), given subcutaneously daily for 5 days, inhibited production of precipitins during immunization of the rabbit with bovine serum albumin. This heterocycle also decreased the number of antibody-forming cells in mouse spleen. ... 

Monomers [180]-15a-d were successfully used for the synthesis of several stereodefined oligo(nucleoside [180]phosphorothioate)s. Their stereochemistry and isotopic enrichment were confirmed by a combined enzymatic-mass spectrometry method employing snake venom phosphodiesterase or calf spleen nuclease, and MALDI TOF mass spectrometry. 

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. ... 

The incorporation of the 5R and 5S diastereoisomers of the 5-hydroxyhydan-toin (derived from oxidative damage of dC) derivative (89) has been achieved using phosphoramidite chemistry and mild deprotection. Whilst the ODNs containing either diastereoisomer were digested with nuclease Pi, the hydantoin derivatives were resistant to digestion by calf spleen and bovine intestinal mucosa phosphodiesterases. 

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