Deoxyribonuclease production

Humidification of inspired air may promote the hydration (liquefaction) of tenacious secretions, allowing for more effective sputum production. The use of mucolytic aerosols (e.g., N-acetylcysteine deoxyribonuclease) is of questionable therapeutic value. Mucolytics may have the greatest benefit... 

Enzymes catalyzing cleavage of DNA, including endo-deoxyribonucleases that generate 5 -phosphomono-esters [EC 3.1.21.x], endodeoxyribonucleases that produce products other than 5 -phosphomonoesters [EC 3.1.22.x], site-specific endodeoxyribonucleases acting on altered bases [EC 3.1.25.x], and exodeoxyribonucleases producing 5 -phosphomonoesters [EC 3.1.11.x]. A few examples are ... 

Deoxyribonuclease I [EC 3.1.21.1], also known as pancreatic DNase and thymonuclease, catalyzes the endonucleolytic cleavage of DNA, preferring dsDNA, to a 5 -phosphodinucleotide and 5 -phosphooligonucleotide end products. 

Deoxyribonuclease II [EC 3.1.22.1], also referred to as pancreatic DNase II, catalyzes the endonucleolytic hydrolysis of DNA (preferring double-stranded DNA) to produce 3 -phosphomononucleotide and 3 -phos-phooligonucleotide end products. 

Deoxyribonuclease (pyrimidine dimer) [EC 3.1.25.1] catalyzes the endonucleolytic hydrolysis of a bond in DNA near pyrimidine dimers to generate products with 5 -phosphates. The enzyme acts on damaged strands of DNA, 5 from the damaged site. 

Two examples where metal ions confer stability or increased activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme ), and Factor VIII. In the case of rhDNase, Ca2+ ions (up to 100 mM) increased the stability of the enzyme through a specific binding site (64). In fact, the removal of calcium ions from the solution with EGTA caused an increase in deamidation and aggregation. However, this effect was observed only with Ca+2 ions other divalent cations, Mg2+, Mn2+, and Zn2+, were observed to destabilize rhDNase. Similar effects were observed in Factor VIII. Ca2+ and Sr2+ ions stabilized the protein, whereas others such as Mg2+, Mn2+ and Zn2+, Cu2+, and Fe2+ destabilized the enzyme (65). In a separate study with Factor VIII, a significant increase in the aggregation rate was observed in the presence of Al3+ ions (66). The authors note that other excipients like buffer salts are often contaminated with Al3+ ions and illustrate the need to use excipients of appropriate quality in formulated products. 

A deoxyribonuclease termed streptodornase, optimally active at pH 7.0 in the presence of magnesium ion, has been partially purified from culture fluids of Streptococcus pyogenes (45). This enzyme yields a distribution of products from DNA very similar to that seen with E. coli endonuclease I Only traces of mono- and dinucleotides are found, the majority of products being rather large oligonucleotides terminated by... 

The reasons for selecting pancreatic DNase I as one of the two representative of mammalian DNases are to a large extent historical. Deoxyribonuclease I was the first enzyme to be recognized as specific for DNA (18-15), the first DNase to produce 5 -monoesterified products (16, 17), the first DNase to be crystallized (18), the first DNase to have a specific protein inhibitor (19-23), the first DNase shown to produce nicks on one strand in preference to scission of both strands (24, 25). A new first has been added recently (25a) DNase I was covalently coupled to porous glass, thus supplying an insoluble DNase. 

Deoxyribonuclease II Porcine spleen Same as DNA as I, except that d bonds are broken and average product is a hexanudeotide... 

Twenty-seven out of 44 FDA-approved biopharmaceuticals have been tested in a battery of genotoxicity assays. Eighty-five different assays performed yielded negative results. The most commonly performed assays were the Ames test, the chromosomal aberration assay in human lymphocytes, the mouse lymphoma gene mutation assay, and the mammalian in vivo erythrocyte micronucleus test. Examples of the range of biopharmaceutical products tested include, domase alfa (deoxyribonuclease I-DNAse), trastuzumab (mAb to human epidermal growth factor receptor 2), alteplase (tissue plasminogen activator), infliximab (mAb to the human tumor necrosis factor a). 

Certain deoxyribonucleases cleave any sequence of single-strand DNA to yield nucleoside monophosphates these enzymes do not hydrolyze base-paired DNA sequences. What products would you expect when you incubate a solution containing a singlestrand specific deoxyribonuclease and the follotving oligodeoxyribonucleotide ... 

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). 

Protein and peptide therapeutics currently represent eight of the top 100 prescription pharmaceuticals in the U.S., and biotechnology products are projected to account for 15% of the total US. prescription drug market by 2003. Of the protein and peptide products now on the market, many are administered as daily injections, though several are delivered by noninvasive routes. For example, desmopressin is delivered as a nasal spray, and deoxyribonuclease I is administered by inhalation. Although cyclosporin A is orally active, as yet there are no general means to confer oral bioavailability to peptides and proteins. A major advance in delivery of peptides was achieved with the introduction of a monthly injectable, biodegradable microsphere formulation of LHRH. 

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. 

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),... 

Miscellaneous.—An e db-j8-D-acetamidodeoxyglucanase obtained from culture filtrates of Streptomyces griseus rapidly released oligosaccharides from Saccharo-myces cerevisiae )8-fructofuranosidase, bovine pancreatic ribonuclease I and deoxyribonuclease 1, and sulphitolysed ovalbumin these glycoproteins contain a D-mannopyranosyl residue attached to di-iV-acetylchitobiose, which, in turn, is linked to an L-asparagyl residue. Analysis of the hydrolysis products showed that the enzyme cleaved between the 2-acetamido-2-deoxy-D-glucopyranosyl residues. 

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