Purification and properties of a magnesium-dependent endodeoxyribonuclease endogenous to rat-liver nuclei.
An Mg2+-dependent endonuclease has been purified from a 0.6 M NaCl extract of rat-liver nuclei by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) was shown to have a pI value of 7.1 and to migrate as a single band to a molecular-weight position of 36,500 on SDS-polyacrylamide gel. The IEF fraction was subjected to a sedimentation analysis. In a hypotonic buffer (10 mM Tris), the nuclease activity sedimented to have an S value of 4.1 S. However, in an isotonic buffer (0.15 M NaCl), this fraction exhibited two activity peaks of 2.8 and 4.3 S. In a hypertonic buffer (0.3 M NaCl), almost all of the nuclease activity sedimented at 2.7-2.8 S. In this connection, values of 2.8 and 4.3 S were determined to correspond to molecular weights of about 36,000 and 70,000, respectively. Thus, an Mg2+-dependent endonuclease, endogenous to rat-liver nuclei, has been inferred to exist in the reversible form of a monomer/homodimer as its native state. Moreover, the IEF fraction formed single-strand nicks more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyguanosine 5'-monophosphate termini in the DNA at an early incubation time. In addition, RNAase activity was not detected in this fraction.
Hibino
Y
Y
Cell Biology Division, Faculty of Pharmaceutical Sciences, Toyama Medical & Pharmaceutical University, Japan.
Yoneda
T
T
Sugano
N
N
D000818:Animals; D002412:Cations; D002467:Cell Nucleus / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology*; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D002412:Cations; D004706:Endodeoxyribonucleases; D008274:Magnesium