Deoxyribonuclease isolation

M. McCarty. Purification anti properties of deoxyribonuclease isolated from beef pftnCttUt JL Col PhysioL 2ft 133-139 (1946). 

Keller and Cohen (36) also subjected to chromatography acidic extracts of cattle pancreatic microsomes and ribosomes. In microsomes they found the expected amounts of all enzymes which are known to be stable in acid, viz., chymotrypsinogen A, trypsinogen, deoxyribonuclease, and ribonuclease. The amounts of chymotrypsinogen B were abnormally low and ribonuclease B was perhaps not present. The results concerning ribosomes were made somewhat uncertain by the ability of these particles to incorporate proteins from the medium. Nevertheless, a series of characteristic enzymes could be isolated from what appears to be the very site of their biosynthesis. 

T.-H. Liao, J. Salnikow, S. Moore, and W. H. Stein. Bovine pancreatic deoxyribonuclease A isolation of cyanogen bromide peptides complete covalent structure of the polypeptide chain. J. Biol. Chem. 248 1489-1495 (1973). [Published erratum appeared in J. Biol. Chem. 267 1951 (1992).]... 

Elenic acid, 7, was isolated from an Indonesian sponge Plakinastrella sp. [25]. It is an enzyme inhibitor of DNA topoisomerase II with an IC50 of 0.1pg/ml [25] and also a potent inhibitor of calf DNA polymerase a and rat DNA polymerases P [250]. Elenic acid was found not to bind to DNA directly and did not affect the activities of plant DNA polymerases I and II, prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes, for example, HIV reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. 

The evidence for independent initiation of mitochondrial DNA synthesis has been described. Do mitochondria utilize the same polymerizing enzymes as nuclei DNA polymerases have been isolated from rat liver mitochondria (Kalf and Ch ih, 1968 Meyer and Simpson, 1968) that manifest all of the requirements shown by the DNA polymerases isolated from bacterial and mammalian cells, but which display marked specificity for mitochondrial DNA as template. Mitochondrial DNA polymerase preparations appeared to be free of nuclear DNA polymerases, terminal addition enzymes, and deoxyribonucleases. These enzymes appear to replicate preferentially native, double-stranded, circular, mitochondrial DNA (Kalf and Qi ih, 1968). These findings strengthen the view that mitochondrial DNA synthesis is a self-contained process. 

During digestive processes, nucleoprotein is split into nucleic acids and protein, the latter then being broken down into amino acids. The nucleic acids are attacked by ribonuclease and deoxyribonuclease enzymes to form nucleotides, which are further hydrolysed by nucleotidases to form nucleosides and phosphates. In the intestines these nucleosides are split by nucleosidases into ribose, deoxy-ribose, purine and pyrimidine bases, which later undergo oxidation and decomposition to ammonia, carbon dioxide and water, to be finally expelled as urea. Nucleotide hydrolysis products are conveniently identified and isolated by chromatographic methods (Chapter 14.2). 

Nickel, Ni a metai present only in traces in living systems. In particular, it seems to be associated with RNA. A nickei metalloprotein, named nickeloplas-min has been isolated from human and rabbit serum, but its function is not known. Ni protects the structure of the ribosome against heat denaturation, and it restores the sedimentation characteristics of E. coli ribosomes that have been denatured by EDTA. Ni can activate some enzymes in vitro, e.g. deoxyribonuclease, acetyl CoA synthetase and phosphoglucomu-tase. Ni deficiency causes changes in the ultrastructure of the liver and alters the level of cholesterol in the liver membranes. It may be important in the regulation of prolactin. 

Changes in chromatin stracture are a hallmark of epigenetic regnlation and developmental plasticity and can be probed on a global scale using the enzyme deoxyribonuclease I (DNase I) to selectively cut open (accessible) chromatin sites (eu-chromatin) in freshly isolated intact liver nuclei (Fig. 11.8a). This technique, known as DNase hypersensitivity site (DHS) analysis, was em-... 

The different procedures for isolating nucleic acids yield products which vary considerably in composition and properties. One reason for this is the presence of nucleolytic enzymes in most plant and animal tissues. Work is always carried out at as low a temperature as possible to retard this enzyme activity and sodium citrate is used in an attempt to inhibit the action of deoxyribonucleases ribonucleases are inactivated with guanidine hydrochloride or dodecylsulphate. 

Deoxyribonucleases. Enzymes that hydrolyze DNA are called deoxyribonucleases (DNAases). In addition to specific enzymes foimd in various extracts, phosphodiesterases such as occur in snake venom hydrolyze DNA. Deoxyribonuclease has been isolated from pancreas.The crystalline enzyme is a protein with a molecular weight around 60,000 and an isoelectric point near pH 5. It requires divalent cations for activity. Like many previously discussed hydrolytic enzymes, it is reversibly denatured by heat. 

Reaction of Deoxyribonuclease. DNAase -acts on polymers of various lengths and forms a mixture of oligonucleotides with very little mononucleotide. Dinucleotides containing purines, pyrimidines, and both have been isolated from digests. The substrate specificity is obviously not so restricted as that of pancreatic RNAase, but has yet to be defined completely. The mechanism of action is also obscure. The linkage of phosphate to the 3 position is split by pancreatic DNAase the monoesters found in the products are all 5 -phosphates. From analogy with ribonucleic acid metabolism, it may be anticipated that DNAase with different specificities will be found, and that some will be found to split the 5 ester bonds. 

Deoxyribonucleic Acid, Since only carbons 3 and 5 of the 2-deoxyribose are available for esterification in DNA, the linkages are all most probably of the 3, 5 -type. Purified phosphodiesterase quantitatively liberates 5 -mononucleotides from thymus DNA 100), On the other hand, no method has as yet been developed for degradation of these nucleic acid to 3 -deoxy-mononucleotides, although the pyrimidine 3, 5 -diphosphates have been isolated from acid hydrolyzates of DNA. Crystalline pancreatic deoxyribonuclease rapidly degrades DNA to very low molecular weight polynucleotides, but identification of many of these products reveals no certain route for the action of the enzyme 168),... 

In 1964, Merrick and Doudoroff isolated PHB granules from B. cereus using lysozymes and deoxyribonuclease to solubilize the peptidoglycans and the nucleic acids respectively. The enzymatic digestion of cell components usually released the nucleic acids in the suspension medirrm which made sirspension highly viscous and impossible... 

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