The radiation-releasable cell wall nuclease of Micrococcus radiodurans. Purification and properties of the native enzyme.
Micrococcus radiodurans is known to possess a surface nuclease located in a mid-wall layer. Previous work showed that hydroxyl radicals, generated by sublethal doses of ionizing radiation, attack the cell wall and initiate release of the active enzyme into the external medium. The enzyme from unirradiated cells has now been purified to homogeneity by a simple 3-step process. The nuclease is a dimer of molecular weight about 260 000 and exonucleolytically degrades both DNA and RNA to 5'-mononucleotides. Single-stranded DNA is degraded at a rate about 200 times faster than double-stranded DNA. Oligonucleotides bearing a terminal 3'-phosphate are resistant to digestion. Dinucleotides must possess a 5'-terminal phosphate and a free 3'-terminal OH to be hydrolyzed. The enzyme has a pH optimum of about 9.0. A metal ion, possibly Ca2+, appears to be tightly bound to the protein. Removal of this metal with EDTA inactivates the enzyme. Simple readdition of Ca2+ does not restore activity although partial function can be recovered by renaturation from urea in the presence of this ion. The availability of pure enzyme should aid in the detection of radiation induced alterations which may be involved in the mechanism of its release from the cell wall.
D002473:Cell Wall / Q000201:enzymology; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology*; D008970:Molecular Weight; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D003851:Deoxyribonucleases; D012260:Ribonucleases