Enzymes deoxyribonucleases

STEREOCHEMICAL TERMINOLOGY, lUPAC RECOMMENDATIONS RESTRICTION ENZYMES DEOXYRIBONUCLEASES Retinoids,... 

Patients with cystic fibrosis use aerosol inhaler sprays of the DNA-hydrolyzing enzyme deoxyribonuclease to help reduce the viscosity of mucous secretions, which contain large amounts of DMA arising from destruction of WBCs as they fight lung Infections. 

M(X)KE. STANFORD (1913-1982). An American biochemist who won the Nobel prize fur chemistry in 1972. with Christian B. Antinscn and William H. Stein, for enzyme studies. He was involved with the analysis ol the action of die complex enzyme deoxyribonuclease. His Ph D. was grunted from the University of Wisconsin. 

Nucleic acid extracted from purified virus using phenol or dodecyl sulfate is easily destioyed by the homologous nucleases present m normal sera or tissues. DNA is destroyed by the enzyme deoxyribonuclease RNA by ribonucleases, This provides one means of identifying the type of nucleic acid. The intact virus is not affected by these enzymes. 

Cystic fibrosis (CF). Patients with CF generally suffer from bacterial infections in the lungs, which is associated with a heavy build-up of thick mucus. Antibiotics are generally prescribed but CF patients also require daily percussion therapy. DNA present in the mucous, which arises from dead white blood cells and bacterial cells, serves to cross link the mucous, changing it from a fluid gel to a semi-solid. The enzyme deoxyribonuclease (DNAase) hydrolyses extracellular DNA, and can be used usefully to alleviate this situation. 

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-... 

Deoxyribonuclease (DNAase), an enzyme that degrades deoxyribonucleic acid, has been used in patients with chronic bronchitis, and found to produce favorable responses presumably by degrading the DNA, contributed by cell nuclei, to inflammatory mucus (213). Lysozyme [9001 -63-2] hydrolyzes the mucopeptides of bacterial cell walls. Accordingly, it has been used as an antibacterial agent, usually in combination with standard antibiotics. Topical apphcations are also useful in the debridement of serious bums, cellulitis, and dermal ulceration. 

Enzyme activity has been sought in seawater Strickland and Solorzano [288] looked for photomono esterase activity, while Maeda and Taga [289] used a flu-orometric method for assaying deoxyribonuclease activity in both seawater and sediment samples. 

Daoust R. Localization of deoxyribonuclease in tissue sections. A new approach to the histochemistry of enzymes. Exp Cell Res 1957 12 203-211. 

Patients suffering from cystic fibrosis often use various aerosolized drugs. To reduce the viscosity of the mucus in the airways, recombinant human deoxyribonuclease is used. This enzyme is the first recombinant protein that has been developed for specific delivery to the lungs via the airways. It has a local action on the mucus in the airways and its absorption is minimal. Another drug that decreases the viscosity of the mucus is acetylcysteine. Aerosolized antibiotics are a further group of therapeutics that is widely used by cystic fibrosis patients. Solutions of antibiotics like tobramycin or colistin are used in nebulizers to prevent exacerbation of the disease. Pentamidine has been used for the prophylaxis of Pneumocystis pneumonia in patients infected with HIV virus, while chronic rejection of lung transplants provided a reason to develop an aerosol formulation of cyclosporine A. 

In addition to the enzymes that catalyse the formation of nucleotides and polynucleotides, a large number of catabolic systems exist which operate at all levels of the internucleotide pathways. The ribonucleases and deoxyribonucleases that degrade polynucleotides are probably not significantly involved in purine analogue metabolism, but the enzymes which dephosphorylate nucleoside 5 -monophosphates are known to attack analogue nucleotides and may be of some importance to their in vivo activity. Phosphatases of low specificity are abundant in many tissues [38], particularly the intestine [29]. Purified mammalian 5-nucleotidases hydrolyse only the nucleoside 5 monophosphates [28] and... 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

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 ... 

Type II site-specific deoxyribonuclease [EC 3.1.21.4], also referred to as type II restriction enzyme, catalyzes the endonucleolytic cleavage of DNA to give specific, double-stranded fragments with terminal 5 -phosphates. Magnesium ions are required as cofactors. 

Deoxyribonuclease X [EC 3.1.22.5] catalyzes the endonucleolytic cleavage of supercoiled plasma DNA to linear DNA duplexes. The enzyme exhibits a preference for supercoiled DNA. 

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. 

The following reviews describe the molecular and physical properties of this broad class of enzymes that catalyze the endohydrolysis of deoxyribonucleic acids and ribonucleic acids. The class includes deoxyribonuclease II [EC 3.1.22.1], Aspergillus deoxyribonuclease Ki [EC 3.1.22.2], deoxyribonuclease V [EC 3.1.22.3], crossover junction endoribonuclease [EC 3.1.22.4], and deoxyribonuclease X [EC 3.1.22.5]. See also Deoxyribonucleases Restriction Enzymes Ribonucleases... 

A second tool employed by the genetic engineer is the enzyme terminal transferase that adds deoxyribonuclease residues to the 3 end of DNA strands creating 3 tails of a single type of residue. 

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. 

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. 

However, in 1944, Avery and associates showed that purified DNA extracted from pneumococci could carry out transformation.8 12 The transforming principal appeared to contain little protein. It was not inactivated by proteolytic enzymes but was inactivated by deoxyribonuclease. Avery was 67 at the time of this discovery, refuting the popular contention that all important scientific discoveries are made by young people. 

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