Purification and properties of DNase gamma from apoptotic rat thymocytes.
We previously identified three distinct DNA endonucleases, DNases alpha, beta and gamma, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, gamma-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase gamma to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase gamma 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 gamma 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 gamma was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase gamma 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 gamma 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 gamma. DNase gamma 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 gamma is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
Shiokawa
D
D
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162, Japan.
Ohyama
H
H
Yamada
T
T
Tanuma
S
S
D000818:Animals; D017209:Apoptosis; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism*; D008297:Male; D009928:Organ Specificity; D051381:Rats; D017208:Rats, Wistar; D013601:T-Lymphocytes / Q000201:enzymology* / Q000473:pathology*
D004706:Endodeoxyribonucleases; C105124:deoxyribonuclease gamma