[Comparative study of Ca/Mg-dependent nucleases from the mammalian thymus]. / Sravnitel'noe issledovanie Ca/Mg-zavisimykh nukleaz timusa mlekopitaiushchikh.

Ca/Mg-dependent nuclease is a possible key enzyme of apoptosis. We isolated and purified nucleases from the human and rat thymus to a homogeneous state and compared some properties of the obtained preparations with those of the earlier isolated Ca/Mg-dependent nuclease from the calf thymus. The activity of the nuclease from the human thymus in the presence of bivalent ions decreases in a sequence:(Ca + Mg) = (Ca + Mn) > Mn, that from the rat thymus: (Ca + Mn) > Mn > (Ca + Mg), and that from the calf thymus: (Ca + Mn) > (Ca + Mg) > Mn. Nuclease are not active in a medium containing only Mg, Ca, Co, and Zn ions. The preparations proved to be unstable during their isolation and storage. If the relative molecular mass of the purifies preparations was according to electrophoresis in 12% DS-Na-polyacrylamide gel 28, 29, and 18.4 and 21 kDa for the calf, human, and rat, respectively, after storage at -20 degrees C for two to six months, the molecular mass of native proteins decreases to 17-14 kDa. Some other properties of the enzymes have been described.

[Cell sensitivity to Fas-mediated apoptosis depends on three forms of Fas-antigen]. / Kletochnaia chuvstvitel'nost' k Fas-oposredovannomu apoptozu zavisit ot trekh form Fas-antigena.

It is known that binding of specific anti-Fas antibodies to Fas-receptor induces apoptosis in various cell lines. But the mechanisms of functional regulation and realization of apoptosis remain so far unknown. We obtained HeLa cells transfected with cDNA of Fas-antigen, whose expression was located under the promoter controlled by isopropoyl beta-D-thiogalactopiranoside. Analysis of transfectants has shown that expression of Fas above a certain critical level leads to redistribution of Fas in the cells and is accompanied by changes in the cell cycles and cell sensitivity to the cytotoxic effects of anti-Fas and tumor necrosis factor (TNF). We propose that the sensitivity of cells to Fas-mediated apoptosis depends on the ratio of transmembrane, intracellular and soluble Fas-antigen in the cells.

Cell cycle specificity of Fas-mediated apoptosis in WIL-2 cells.

Antibodies to Fas/APO1 receptor induce effective apoptosis in WIL-2 cells of the human B-lymphoid line. Quantitative assessment of the extent of the death in cells synchronized by thymidine block revealed a significant increase in their sensitivity to the cytocidal effect mediated by Fas/APO1 during the G1 phase of the cell cycle. Western analysis of the content of the p53 antigen in the cytoplasm and nuclei of the cells showed that the Fas/APO1-induced death is accompanied by massive translocation of the p53 from the cytoplasm to the nucleus. These findings suggest that cell vulnerability to the Fas/APO1-mediated apoptosis is subjected to regulation by cell cycle-dependent mechanisms, one of which is probably the function of the p53 antigen.

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

Three nuclease activities have been found and characterized in rat thymocyte nuclear extracts. A Mn(2+)-dependent nuclease is loosely bound to nuclear components and can be extracted with 0.35 M NaCl. The enzyme is activated by Mn2+ but not by Mg2+, Ca2+, or both. Its molecular mass is 36-40 kDa when measured by gel filtration and 37 kDa by SDS/PAGE. 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+/Mn(2+)-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 Mg2+ or Ca2+ but cleave DNA in Ca2+/Mg(2+)-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+/Mn(2+)-dependent nuclease from thymocyte nuclei separated by chromatography on CM-Sepharose and does not change the activities of Mn(2+)-dependent and acidic nucleases. Nuclease activity in thymocyte nuclei from irradiated rats is increased in Ca2+/Mg(2+)-containing and Ca2+/Mn(2+)-containing media whereas there is no change in the activity of acidic nuclease. Ca2+/Mn(2+)-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 Ca2+/Mn(2+)-dependent-nuclease binding to the nuclear structures in dying thymocytes are discussed.

[Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin]. / Vydelenie Ca2+, Mg2+-zavisimoi nukleazy iz khromatina timusa telenka.

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.

On the role of Ca, Mg-dependent nuclease in the postirradiation degradation of chromatin in lymphoid tissues.

The characteristics of the postirradiation degradation of chromatin in thymocytes in vivo were compared with the features of chromatin fragmentation in isolated thymocyte nuclei in vitro by endogenous chromatin-bound nucleases. Nuclease which degrades chromatin produces in vivo fragments of nucleosomal size; the double-strand breaks appear as the result of the accumulation of single-strand breaks with 3'-OH ends; the nuclease is inhibited by Zn2+ and DTNB and its activity is depressed by cycloheximide pretreatment. In experiments on in vitro degradation of chromatin in isolated thymocyte nuclei similar properties were observed for the Ca, Mg-dependent, but not for acid nuclease. The results bring further evidence of the involvement of an enzyme of the Ca, Mg-dependent nuclease-type in chromatin degradation in irradiated thymocytes.

Inhibition of poly(ADP-ribose) polymerase as a possible reason for activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats.

The molecular mechanism of activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats was studied. Thymocyte nuclei of control and irradiated rats were pre-incubated with NAD under conditions favourable for poly ADP-ribosylation. Pre-incubation results in a decrease in the rate of autolytic DNA digestion by Ca2+/Mg2+-dependent endonuclease of 6-7- and 2-3-fold for control and irradiated animals, respectively. The activity of Ca2+/Mg2+-nuclease extracted from the nuclei pre-incubated with NAD is also considerably decreased. The presence of nicotinamide and thymidine in the preincubation medium prevents the suppression of Ca2+/Mg2+-nuclease activity. In the experiments performed with isolated nuclei and permeabilized thymocytes the synthesis of poly(ADP-ribose) does not significantly change within 1 h after irradiation at a dose of 10 Gy, whereas 2 and 3 h after the exposure it decreases by 35-40 and 45-55 per cent, respectively. The activity of poly(ADP-ribose) glycohydrolase in this period is similar to that in the controls. The average size of the de novo synthesized chains of poly(ADP-ribose) increases from 11 to 17 ADP-ribose units by the second hour after irradiation. Inhibition of poly(ADP-ribose) polymerase in the postirradiation period preceded the internucleosomal fragmentation of chromatin. The results suggest that activation of Ca2+/Mg2+-nuclease in irradiated thymocytes is accounted for by the disturbance of its poly ADP-ribosylation.

[Postradiation inhibition of poly ADP ribose polymerase is not determined by internucleosomal fragmentation of DNA]. / Postradiatsionnoe ingibirovanie poli(ADF-ribozo)-polimerazy ne opredeliaetsia mezhnukleosomnoi fragmentatsiei DNK.

Postirradiation changes in poly(ADP-ribose) polymerase activity in nuclei and permeable thymocytes were studied. The incorporation of 14C-NAD into poly(ADP-ribose) was virtually invariable during the first 60 min after irradiation at a dose of 10 Gy, but after 2 and 3 h it made up, as compared to the control, 60 and 45% for nuclei, and 65 and 55% for permeable thymocytes, respectively. It was shown that the internucleosomal fragmentation of DNA was not responsible for the observed changes in poly(ADP-ribose) polymerase activity.

The involvement of nuclear nucleases in rat thymocyte DNA degradation after gamma-irradiation.

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes--a Ca2+/Mg2+-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca2+/Mg2+-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca2+/Mg2+-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg2+-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca2+/Mg2+-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes.

[Characteristics of acid ribonuclease from rat thymus chromatin]. / Kharakteristika kisloi ribonukleazy khromatina timusa krysy.

Acid ribonuclease, free of nucleases and phosphatases, is isolated from rat thymus chromatin. The pH optimum of the enzyme is 5.0-5.5, optimal concentrations of Na+ and K+ ions are 0.05-0.15 M and 0.05 M respectively, Mg2+ inhibits the enzyme activity. The enzyme hydrolyses poly U, poly AU, cytoplasmic and nuclear RNAs, but does not attack poly A, polyG, polyC, poly A:poly U, native and denatured DNA'S. The enzyme is 3'-endonuclease, it splits the bond between the 5'-carbon atom of adenosine, guanosine and uridine and 3'-phosphate of uridilic residue. Middle length of oligonucleotides after the hydrolysis of cytoplasmic RNA comprises 10 nucleotides. Possible role of the enzyme in the processing of nuclear RNAs is discussed.