We previously identified three distinct DNA endonucleases, DNases α, β and γ, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, γ-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase γ to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase γ exhibited one predominant protein band on SDS/PAGE and an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase γ was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine and lysine residues. The N-terminal amino acid sequence of DNase γ was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase γ requires both Ca2+ and Mg2+ for activity. This requirement could be partially supplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ and Zn2+ inhibited DNase γ activity. These bivalent cations also suppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X-rays. The same order of inhibitory ability was observed for these bivalent metal ions in vivo(in intact cells) and in vitro, suggesting that the suppression of apoptotic DNA fragmentation at the cellular level is due to the inhibition of DNase γ. DNase γ activity was found to exist at high levels in spleen, lymph node, thymus, liver and kidney, but little was present in brain, heart or pancreas. On the basis of these findings, together with previous data, we conclude that DNase γ is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
Research Article|September 15 1997
Purification and properties of DNase γ from apoptotic rat thymocytes
*Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162, Japan
§Research Institute for Bioscience, Science University of Tokyo, Noda, Chiba, Japan
1To whom correspondence should be addressed.
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Daisuke SHIOKAWA, Harumi OHYAMA, Takeshi YAMADA, Sei-ichi TANUMA; Purification and properties of DNase γ from apoptotic rat thymocytes. Biochem J 15 September 1997; 326 (3): 675–681. doi: https://doi.org/10.1042/bj3260675
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