Pancreatic nucleases

In common with the digestion of other macromolecules, nucleic acids are hydrolysed in a stepwise manner, by pancreatic nuclease (diesterase enzymes) which hydrolyse the bonds between two adjacent phosphate groups in RNA and DNA. The resultant oligoribonucleotides and oligode-oxy ribonucleotides are hydrolysed to form nucleoside monophosphates, which lose their phosphate to form nucleosides, by the action of pancreatic phosphatase. In brief, the process is ... 

The intestine also contains enzymes capable of digesting the nucleic acids—ribcoucleic acid (RNA) and deoxyribonucleic acid (DNA). Pancreatic nucleases split nucleic acids into nucleotides (purine or pyrimidine base, sugar, and phosphoric acid), and the nucleotides are split into nucleosides (purine or pyrimidine base and a sugar), and phospharic acid. These nucleosides are then split into their constituent sugar (pentoses), purine (adenine or guanine) and pyrimidine ( osine, uracil, or thymine) bases. These bases are then absorbed by active transport... 

FIGURE 11.32 All example of nuclease specificity The specificity of RNA hydrolysis by bovine pancreatic RNase. This RNase cleaves h at 3 -pyriinidines, yielding oligonncleoddes with pyrimidine 3 -P04 ends. 

Hydrolysis of RNA by alkali or pancreatic RNase leads initially to fragments which terminate in 2, 3 -cyclic phosphodiesters. Micrococcal nuclease, on the other hand, gives rise to fragments terminating in 3 -phos-phomonoester groups which facilitate their isolation, and this enzymic hydrolysis has been used to prepare 3 -ribodinucleotides. ... 

If polyribonucleotides are treated simultaneously with methoxylamine and bisulphite, cytidine residues are converted into 5,6-dihydro-7V4-methoxycytidine-6-sulphonate,154 and uridine into 5,6-dihydrouridine-6-sulphonate.155 Treatment with dilute ammonia regenerates the uridine residues, leaving the dihydrocytidine derivatives unaffected. When only the cytidine residues have been derivatized, pancreatic ribonuclease becomes uridyl ribonuclease, since it is unable to cleave the chain on the 3 -side of the modified cytidine.154 This allows the isolation of blocks of modified cytidine residues. T2 ribonuclease may also be used. Alternatively, a ribonuclease from Physarum polycephalum has been found to hydrolyse CpX links very slowly, allowing the isolation of cytidine blocks.156 If both uridine and cytidine residues are modified, T2 ribonuclease acts as puryl ribonuclease, allowing the isolation of cumulative blocks of pyrimidines.155 This ability to alter the specificity of nuclease cleavage is a useful tool in sequence analysis. 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Hexokinase Pyruvate kinase Adenylate kinase Phosphoglycerate kinase Phosphofructokinase Protease inhibitors Pancreatic trypsin inhibitor Soybean trypsin inhibitor Streptomyces subtilisin inhibitor Nucleases... 

Pancreatic ribonuclease Staphylococcal nuclease Peroxidases Glutathione peroxidase Cytochrome c peroxidase Oxygen carriers Myoglobin, hemoglobin Myohemerythrin, hemerythrin Hormone-binding proteins Uteroglobin Pre albumin Lectins... 

The mechanism involves participation of the free 2 -OH of the ribose groups and formation of cyclic 2, 3 -phosphates and is similar to that of pancreatic ribo-nuclease (Chapter 12). Because deoxyribose lacks the free 2 -OH, the phosphodiester linkages in DNA are quite stable in base. 

A multitude of nucleases cleave DNA, single- or double-stranded. They range from the pancreatic digestive enzyme DNase I through specialized nucleases that function during DNA repair and the hundreds of restriction endonucleases that have become so valuable in modern laboratory work. Some nucleases leave a 3 -phosphate ester at a cut end in a DNA chain, while others leave a 5 -phosphate end.824 Many nucleases are dealt with in later chapters. Only a few will be mentioned here. 

Fig. 3. Denaturational increment of the partial specific heat capacity of pancreatic ribo-nuclease A (RNase), hen egg-white lysozyme (Lys), and sperm whale myoglobin (Mb). The dashed lines represent the parts of these functions that were obtained by a linear extrapolation of th partial heat capacity of the native state. The dot-and-dash lines show the behavior when the values measured at 50°C are assumed to be temperature independent.

Abstract The major enzymatic barrier to the absorption of macromolecules, particularly therapeutic peptides, is the pancreatic enzymes the peptidases, nucleases, lipases and esterases that are secreted in considerable quantities into the intestinal lumen and rapidly hydrolyse macromolecules and lipids. In the case of the peptidases, they work in a co-ordinated fashion, whereby the action of the pancreatic enzymes is augmented by those in the brush borders of the intestinal cells. The sloughing-off of mucosal cells into the lumen also furnishes a mixture of enzymes that are a threat to macromolecules. As the specificity and activity of the enzymes are not always predictable, during pharmaceutical development it is important to test the stability of therapeutic macromolecules, and novel macromolecular-containing or lipid-containing formulations, in the presence of mixtures of pancreatic enzymes and bile salts, or in animal intestinal washouts or ideally, aspirates of human intestinal contents. 

The other major enzymes in pancreatic juice of relevance to our discussion are the nucleases. Human pancreatic DNase I has a molecular weight of around 30 kDa, shows maximal activity in the pH range 7.2-7.6 and requires metal ions such as Mn+, Mg+ or Co+ in the presence of Ca+. It is an endonuclease that hydrolyses the phosphodiester linkages in both single- and double-stranded DNA at multiple sites (Funakoshi et al. 1977 Takeshita et al. 2000). 

The other group of enzymes of major concern to our discussion are the nucleases secreted by the pancreas, which are very much in the minority as they constitute less than 1% of the pancreatic juice protein (Scheele et al. 1981). Again this is in quantitative protein terms, and I can find no information concerning their actual activity in the lumen of the intestine. 

Q7 Pancreatic exocrine tissue produces amylase, lipase and a range of serine proteases, enzymes such as trypsin (which is also elevated in pancreatitis), chy-motrypsin and elastase, also nucleases, carboxypeptidase and aminopeptidase. 

Many other ribonucleases have been described. They differ with respect to specificity toward various internucleotide bonds, in heat stabilities, and in pH optima. In 1949 Maver and Greco (M8) reported the presence in extracts of spleen of a heat-labile nuclease that had a pH optimum of approximately 5.2 in the presence of magnesium, and 6.6 in the absence of magnesium. That this was a different enzyme entirely from pancreatic RNase was shown most dramatically by Hilmoe and Heppel (H7), who found that this enzyme was able to hydrolyze the limit polynucleotide obtained by exhaustive treatment of RNA with ribonuclease I. The ubiquity of ribonucleases can perhaps best be demonstrated by noting... 

A. J. Doherty, A. F. Worrall, and B. A. Connolly. Mutagenesis of the DNA binding residues in bovine pancreatic DNase 1 An investigation into the mechanism of sequence discrimination by a sequence selective nuclease Nucleic Acids Res. 19 6129-6132(1991). 

The nudeo-proteins under the influence of enzymes are successively transformed into nucleins, into nudeic adds, then into xanthin bases, into phosphoric acid, etc. But only the first phase of the transformation, that which corresponds to the formation of protein and nudein, can be realized by p>epsin and trypsin. The nudeins resist the action of the digestive juices, botii gastric arid pancreatic. The transformation of the nucleins first into nucldc adds, then into phosphoric add and into purin bas, is induced in living cells, on the contrary, by the endo-cellular enzymes, which bear the name of nucleases. 

Preparations of pancreatic ribonuclease, ribonuclease from Actinomyces rimosus, and the nuclease from Serratia marcescens have been covalently bound to 4 3-hydroxyethylsulphonylanisole-, 4j3-hydroxyethylsulphonylaniline, and 3-Cl-2-hydroxypropyl derivatives of dextran and dialdehyde-dextran and to the 4-aminobenzyloxymethyl ester of dextran.The yield and thermal stability of the modified nucleases depends both on the amount and the character of the functional groups activating the polysaccharide. 

Pancreatic ribonuclease, ribonuclease from Actinomyces rimosus, and the nuclease from Serratia marcescens have been covalently bound to the 3-amino-benzyloxymethyl ester of D-mannan. ... 

Polynucleosomes were isolated from purified rat pancreatic nuclei digested by micrococcal nuclease to an acid solubility of 3 to 5% as previously described [4,11]. They were poly(ADP-ribosylated by purified calf thymus DNA-bound poly(ADP-ribose) polymerase [14], using a ratio of 3 /ug of the purified enzyme to 1 A260 unit of polynucleosomes and a NAD concentration of 200 juAf under the same conditions as... 

---