Deoxyribonuclease substrate

Amylases, peptidases and deoxyribonuclease mobilize many nutrients that are released from lysed cells. They also deerease the viseosity of fluids present at the lesion by depolymerization of their biopolymer substrates. 

The TNF-a-induced DNA fragmentation may have been caused by caspase-activated deoxyribonuclease (CAD) in PC-12 cells. In non-apoptotic cells, CAD is present as an inactive complex with the inhibitor jOvd p3,24]. During apoptosis, caspase-3 inactivates ICAD, leaving CAD free to function as a nuclease [25]. Therefore, we used die fluorogenic substrate, Ac-DEVD-MCA, to determine whether the caspase-3 in PC-12 cells was activated by treatment with TNF-a and/or crocin. As shown in... 

The nucleases are enzymes that hydrolyse nucleic acids, either deoxyribonucleases (DNases) that have DNA as the substrate or ribonucleases (RNases) that have ribonucleic acids as the substrate. The DNases hydrolyse the phosphodiester linkages between the deoxyribose molecules of DNA, and similarly, the RNases attack the equivalent bonds in RNA. There are many nucleases found in mammalian tissues, and as in the case of the peptidases, they can be divided into the categories endo and exo based on whether they attack bonds in the interior of the nucleic acid molecule or remove nucleosides from the end termini of the chains. They... 

Fig. 5. The execution phase of apoptosis. An apoptotic stimulus causes the release of cytochrome c from mitochondria. The first box contains the components required to activate caspase 9 card refers to the caspase-recruitment domain. Caspase 9 then activates caspase 3 (second box), which in turn activates caspase 6 (last box). The amino-acid sequences at the cleavage sites are shown. Caspases 3 and 6 also have a prodomain that is not present in the active protease in caspase 6 an additional cleavage removes a small portion of the middle of the protein. Various substrates of the caspases are shown, including the pathway by which caspase-activated deoxyribonuclease (CAD) is activated which in turn leads to DNA...

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. 

In the body, enzymes are compartmentalized and function under highly restricted conditions. Some enzymes (e.g., proteinases) are not substrate-specific. When present in active form in an inappropriate part of the body, they act indiscriminately and cause considerable damage to the tissue. Inhibitors inactivate these enzymes at sites where their action is not desired. Proteinase inhibitors, which are themselves proteins, are widely distributed in intracellular and extracellular fluids. Protein inhibitors of enzymes other than proteinases are relatively rare. Such inhibitors are available for a-amylases, deoxyribonuclease I, phospholipase A, and protein kinases. 

D3. Daoust, R., and Amano, H., Ribonuclease and deoxyribonuclease activities in experimental and human tumors on the histochemical substrate film method. Cancer Res. 28, 131-134 (1963). 

T.-H. Liao, H.-C. Ho, and A. Abe. Chemical modification of bovine pancreatic deoxyribonuclease with phenylglyoxal—the involvement of Arg-9 and Arg-41 in substrate binding. Biochim. Biophys. Acta 1079 335-342 (1991). 

Whether the interaction between histone and DNA is specific and occurs in vivo to repress DNA is not certain. However, it cannot be excluded that the histones interfere with the polymerase reaction simply by making the primer unavailable in a fashion common to all basic polymers. Histones and protamines interfere with the hydrolysis of DNA by deoxyribonuclease, and it thus can be established that the interference results from the precipitation of substrate. Injected, histones have been claimed to interfere with DNA biosynthesis in regenerating liver. Such intriguing observations undoubtedly have to be confirmed in several laboratories before their full impact can be appreciated. In conclusion, modern studies suggest that histones may be important in the transfer of genetic information from DNA to messenger RNA, but the molecular details remain to be elucidated. 

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. 

A kinetic study on the activation of deoxyribonuclease I by magnesium has shown that the activation curve is biphasic (the substrate being the Mg salt of bovine spleen DNA). This indicates that activation occurs at two sites on the protein. Free Mg + was required for enzyme activity, confirming that a metallo-enzyme as well as a metallo-substrate is necessary for deoxyribonuclease I activity. In contrast, it has been shown that the pyrophosphatase activity of bovine brain alkaline phosphatase depends on Mg + bound to the enzyme but not on the formation of a magnesium-substrate complex. 

---