Properties of some nuclear nucleases of rat thymocytes and their changes in radiation‐induced apoptosis

Abstract

Three nuclease activites have been found and characterized in rat thymocyte nuclear extracts. A Mn2+-dependent nuclease is loosely bound to nuclear components and can be extracted with 0.35 M NaCI. The enzyme is activated by MnZ+ but not by Mg", Ca2+, or both. Its molecular mass is 36-40 kDa when measured by gel filtration and 37 kDa by SDSPAGE. An acidic nuclease is independent of divalent ions, produces DNA strand breaks with 5'-OH ends, its molecular mass is about 37 kDa. Two fractions of Ca2+Nn2+-dependent nuclease, differing in binding to CM-Sepharose but identical in other respects, are active in the presence of Mn2+ but can be additionally activated by Ca2+. They are inactive in the presence of Mg'' or Ca2+ but cleave DNA in Ca2'/Mg2'-containing medium. The molecular mass of the enzyme is 22 kDa as determined by both gel filtration and electrophoresis. The dependence of nuclease activities on pH, ions, and sulfhydryl reagents is described. Cycloheximide injection to both control and irradiated animals strongly inhibits the activities of Ca2+/Mn2+-dependent nuclease from thymocyte nuclei separated by chromatography on CMSepharose and does not change the activities of Mn2+-dependent and acidic nucleases. Nuclease activity in thymocyte nuclei from irradiated rats is increased in CaZ+Ng2'-containing and Ca2+/ Mn"-containing media whereas there is no change in the activity of acidic nuclease. CaZ+/Mn2+-dependent nuclease is extracted from thymocyte nuclei of irradiated rats with 0.35 M NaCl but from control nuclei only with 0.5 M NaCl. Possible reasons of labilization of Ca*+Nn*+-dependentnuclease binding to the nuclear structures in dying thymocytes are discussed.In the past decade the concept has been formulated that in most cases the death of cells is the result of their active response to physiological or damaging factors [ 1 -31. This type of cell death was called apoptosis [l] and the recent literatur data suggests that, besides the programmed and physiological cell death [l -31, cell death [5-81. Data indicate the involvement of nuclear nucleases, particularly, Ca2+Ng2+-dependent endonuclease [7, 21 -241 but the mechanism of itdtheir activation in dying cells remain unclear. A number of preparations of this enzyme were purified from different sources but they usually differed in molecular mass, substrate specificity, binding to ion-exchange columns and so on [25-301. Ca2+/Mg2+-nucleases with molecular masses in the range 25-120 kDa with preference for single-stranded or double-stranded DNA are described. The mechanisms of regulation of the nuclease activity are poorly understood. Even the ion dependence of the nuclease differs between various studies. The enzyme is normally inactive in the presence of Mg2+ or Ca" only but is activated by addition of both ions. At the same time the nuclease is normally active in the presence of Mn2+ and the maximal activity is manifested with Mn' + and Caz+ [25, 301. Previously it has been shown that there are different nucleases in rat thymus nuclei but only a CaZ...

Detection of Higher-Order 50- and 10-kbp DNA Fragments before Apoptotic Internucleosomal Cleavage after Transient Cerebral Ischemia

“…In the final analysis, no overall acceptable degradative scheme has been found, and different cells might use different cleaving activities to disassemble the genome when entering an apop totic cell death pathway. What does appear to be accepted is that only 3' -OH end groups are generated in the laddered DNA in dying cells (Alnemri and Litwack, 1990;Nikonova et at., 1993). This available hydroxyl group permits end-labeling by TdT and has led to wide use and commercialization of the TdT mediated dUTP-biotin nick end labeling (TUNEL) technique (Gavrieli et at., 1992) for in situ identifica tion of DNA fragments in dying cells and tissue sec tions.…”

Induction of Apoptosis in Fish Cells by Hypertonic Stress

“…[21][22][23][24][25][26][27] In EPC, the DNA fragmentation induced by hypertonic stresses (both 450 and 500 mOsm/kg) as well as by heat shock, UV irradiation, and actinomycin D was partially suppressed by 1 mm Zn2+ ion (Fig. 6).…”

Multi‐step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents

“…These results, coupled with the findings with Cd 2+ and Hg 2+ which are classical sulphydryl inhibitors, strongly suggest that a critical thiol is involved in the DNA fragmentation process. The thiol group may be located on the endonuclease itself as Nikonova et al [28] have reported that three nucleases extracted from thymocyte nuclei were inhibited by NEM and idoacetamide. Also, Ribeiro and Carson [29] have described the purification of a CaZ+/Mg 2+ endonuclease from human spleen, which was partially inhibited by NEM.…”