[Effect of gamma-radiation on the activity of proteases associated with spleen and brain nuclear histones of young and old rats].

It has been found that proteases specifically splitting histones are associated with histones from spleen and brain nuclei of 4- and 26-month-old rats. The activity ofproteases isolated together with histones increases after irradiation of rats with 10 Gy The activation degree of these proteases depends on the animal age and postradiation period. Activation ofhistone-associated proteases by means of gamma-radiation is more pronounced in spleen nuclei from old rats than from the young ones. Irradiation of animals has been found to reduce histone H1 and core histone contents in the spleen and brain nuclei of both young and old rats. The radiation-induced proteolysis ofhistone H1 and core histones in spleen and brain nuclei leads to chromatin deconden-sation and DNA degradation by nucleases. The activity of histone-associated proteases is substantially higher in the nuclei of intensively proliferating spleen cells than in the brain nuclei. The experimental data indicate that histone-associated proteases participate in the regulation of DNA transcription, replication, and degradation.

[Study of DNA-binding proteins in mitochondria of rat liver gamma-irradiation].

Acid-soluble proteins were isolated from the liver mitochondria of control and irradiated (8 Gy) rats. By means of electrophoresis in 15% polyacrylamide gel, these proteins were separated into more than 20 polypeptides of molecular masses between 10 and 120 kDa. The irradiation of rats with a dose of 8 Gy led to changes in the polypeptide content of mitochondrial acid-soluble proteins in the postradiation period. It was found that the liver acid-soluble proteins of control and irradiated rats were able to form nucleoproteid complexes with DNA at the physiological NaCl concentration. It was shown that along with mitochondrial acid-soluble proteins, proteases were also released, their activity increased in the presence of DNA. Twenty four hours after irradiation of rats with 8 Gy, the activity of proteases cleaving mitochondrial acid-soluble proteins decreased. Probably, the acid-soluble proteins and DNA-activated proteases of mitochondria are involved in the regulation of the structural organization and functional activity of mitochondrial DNA.

[Study of DNA-binding proteins of rat liver mitochondria].

Acid-soluble proteins able to form DNA-protein complexes in the presence of physiological concentration of NaCl were isolated from rat liver mitochondria. Electrophoretic analysis of these proteins in 15% polyacrylamide gel showed that mitochondrial acid-soluble proteins include of approximately 20 polypeptides with molecular weight of 10-120 kDa. The fraction of acid-soluble proteins can be separated into basic and acidic proteins by chromatography on DEAE cellulose. Some of acidic proteins are tightly bound to the basic proteins and can be separated from them in the presence of 5 mM dithiothreitol. It is discovered that the fraction of acidic proteins contains proteases (including DNA-activated ones), which cleave different polypeptides of the basic proteins with different efficiency. Possibly, mitochondrial DNA-binding proteins and DNA-activated proteases are involved in the regulation of structural organization and functional activity of mitochondrial DNA.

Study of protein carbonyls in subcellular fractions isolated from liver and spleen of old and gamma-irradiated rats.

Age- and gamma-irradiation-dependent accumulation of oxidatively modified proteins (measured as carbonyl level) was studied in cytoplasm, mitochondria and nuclei isolated from spleen and liver of 4- and 26-month-old rats. The protein carbonyl levels significantly increased with age in all fractions studied. The carbonyl content was found to be two times higher in the nuclei than in the mitochondria and cytoplasm, which may be related to an extensive modification of lysine and arginine residues in histone molecules. Gamma-Irradiation of rats with 10 Gy caused a rise of protein carbonyls only in their cytoplasm and mitochondria, which was prevented in the animals fed with antioxidants and vitamins for a month before the irradiation. We observed an activation of histone-specific proteases in the nuclei of gamma-irradiated rats. The lack of carbonyl accumulation in the nuclear proteins isolated from tissues of gamma-irradiated animals may be explained by the degradation of oxidized histones by these proteases.

Gamma irradiation or hydrocortisone treatment of rats increases the proteinase activity associated with histones of thymus nuclei.

An increase in the activity of histone-associated rat thymus nucleus proteinases specific for histones H2A, H2B and H1 was shown after gamma irradiation or hydrocortisone treatment of animals. Histone H1-specific proteinase activity is dependent on DNA and increases in the presence of denatured DNA, whereas proteinases specific for core histones are inhibited in the presence of denatured DNA. The increase in the activity of histone-associated proteinases depends on the radiation dose and the time after irradiation or hydrocortisone injection. In the presence of dithiotreitol and sodium dodecyl sulfate, these proteinases dissociate from histones. It was found by gel electrophoresis that several proteinases of various molecular masses are closely associated with histones obtained from thymus nuclei of irradiated or hydrocortisone-treated rats.

Gamma-irradiated DNA activates histone H1-specific proteinase of rat liver nuclei.

We investigated the effect of various forms of DNA (double- and single-stranded calf thymus DNA, circular plasmid DNA, gamma- and UV-irradiated DNA and DNAase I-treated double-stranded DNA) aggregated with histones, on the proteolysis of these histones by proteinase associated with the rat liver nuclear scaffold. It was shown that the nuclear scaffold-associated proteinase is able to degrade selectively the histone H1 only in the presence of the DNA containing single-strand breaks induced by gamma-radiation or DNAase I treatment as well as in the presence of heat-denatured DNA. This proteinase is not activated by the double-stranded circular plasmid DNA or by UV-treated double-stranded DNA. Histone H1-specific proteinase (HSP) activated by gamma-irradiated DNA is inhibited by inhibitors of serine proteinases such as antipain, leupeptin, phenylmethylsulphonyl fluoride, as well as by dithiothreitol. The results lead us to suggest that DNA-activated HSP from rat liver nuclei is involved in the regulation of the access of repair enzymes to the damage portions of DNA within chromatin.

[DNA and nucleotide triphosphate-activated proteinase from rat liver nuclear matrix specific for H1 histone]. / Aktiviruemaia DNK i nukleotidtrifosfatami proteinaza iadernogo matriksa pecheni krysy, spetsifichnaia k gistonu H1]

The action of DNA and nucleotide phosphate on histone hydrolysis by nuclear matrix preparations from rat liver has been studied. It is shown that proteinase specific for H1 histone is associated with the nuclear matrix. This proteinase is activated by denatured DNA and by DNA treatment with DNase I or gamma-irradiation, but it is not activated by UV-irradiated DNA. In the presence of nucleotide triphosphates, particularly GTP and ATP, proteolysis of H1 histone is markedly increased. The nuclear matrix proteinase specific for H1 histone and activated by DNA or GTP and ATP appears inhibited by antipain, leupeptin, phenylmethylsulfonyl fluoride (the inhibitors of serine proteinases) as well as by dithiotreitol.

[Association of proteinases with histones from rat thymus nuclei]. / Assotsiatsiia proteinaz s gistonami iader timusa krys.

Histone H2A, H2B, and H1--specific proteinases tightly associated with histones were shown to be present in rat thymus nuclei. The activity of proteinases tightly associated with histones increases after exposure of animals to gamma-rays. The denatured DNA activated the histone H1-specific proteinase. These proteinase dissociated from histones in the presence of dithiothreitol. The histones and proteinases were divided into fractions by chromatography on DEAE-cellulose in the presence of 5 mM dithiothreitol.

[Effect of gamma-radiation and mitochondrial apoptogenic factors on nuclear protease activity]. / Vliianie gamma-radiatasii i mitokhonkrial'nykh apoptogennykh faktorov na aktivnost' iadernykh proteaz.

An increase in protease activity was shown in thymus nuclei of rats exposed to gamma-radiation. The activation of histone-specific proteases depended on the duration of postradiation period. Also, it was revealed that incubation of thymus nuclear with the intermembrane fraction of liver mitochondria caused degradation of histones and nonhistone nuclear proteins, as well as internucleosomal fragmentation of DNA. Simultaneously, nuclear proteases tightly bound to histones and specifically cleaving histones were observed to be activated by apoptogenic factors of the mitochondrial intermembrane fraction. Probably, the apoptogenic action of gamma-radiation involves not only a direct DNA damage that induces activation of DNA-dependent proteases but also an indirect component: destructive alterations in mitochondria leading to the exit of apoptogenic factors from the intermembrane space.

[Change in the initiation of DNA synthesis on the nuclear matrix and its DNA-protein composition in gamma-irradiated hepatoma cells]. / Izmenenie initsiatsii sinteza DNK na iadernom matrikse i ego DNK-belkogo sostava v gamma-obluchennykh kletkakh gepatomy.

It was shown that gamma-irradiation of Zajdela hepatoma cells (10 Gy) induces inhibition of DNA synthesis initiation at a nuclear matrix and a change in its DNA-protein content. Irradiation of hepatoma cells with 10 and 50 Gy decreases incorporation of newly synthesized proteins in the firmly bound DNA-protein complexes of nuclear matrix. After 60-120 min postirradiation incubation of cells at 37 degrees C DNA-protein content of the nuclear matrix and its firmly bound DNA-protein complexes are restored. However the rate of DNA synthesis initiation being below the control level.

[Reorganization of the protein composition of the nuclear matrix of hepatoma cells after inhibition of DNA synthesis with novobiocin]. / Issledovanie reorganizatsii belkovogo sostava iadernogo matriksa kletok gepatomy pri ingibirovanii sinteza DNK novobiotsinom.

The nuclear matrix of Zajdela hepatoma cells, in which DNA synthesis was blocked by novobiocin, contained 2.5-3.0 times more DNA and protein not dissociating in 2 M NaCl than the nuclear matrix of control cells. Chromatography of nuclear matrix preparations on Sepharose 2B-CL resulted in isolation of tightly bound DNA-protein complexes which did not dissociate in 8 M urea or 0.1% SDS. Subsequent elution of DNA-protein complexes on a hydroxylapatite column with a buffer containing 4 M guanidine hydrochloride and 5 M urea caused partial dissociation of the complexes. Electrophoretic analysis revealed essential changes in the composition of proteins DNA-protein complexes of hepatoma cells nuclear matrix during inhibition of DNA synthesis.

[Postradiation activation of proteinases associated with the hepatocyte nuclear matrix in rats]. / Postradiatsionnaia aktivatsiia proteinaz, assotsiirovannykh s iadernym matriksom gepatotsitov krys.

Proteinase activity of the nuclear matrix of rat hepatocytes was 7-8-times as high as that of initial nuclei. Activity of nuclear matrix proteinases was optimum at pH 8-9. Proteolytic activity associated with the nuclear matrix, increased by 1.4-2.8 times 2 h following irradiation with doses from 5 to 30 Gy. Cycloheximide, a protein synthesis inhibitor, administered to animals failed to suppress the radiation-induced increase of proteinase activity of the nuclear matrix.

[Hydrocortisone and partial hepatectomy activate a proteinase associated with histones]. / Gidrokortizon i chastichnaia gepatéktomiia aktiviruiut proteinazu, assotsirovannuiu s gistonami.

Proteinase activity has been shown to be associated with histones of rat thymus and liver nuclei. Hydrocortisone increases the activity of proteinases associated with thymus nuclear histones. Increasing activity of histone-associated proteinases is also observed during intensive transcription and replication in regenerating rat liver.

[Protease activity of the nuclear matrix of rat hepatocytes]. / Proteaznaia aktivnost' iadernogo matriksa gepatotsitov krys.

It was demonstrated that the nuclear matrix of rat liver possesses the protease activity. The specific activity of nuclear matrix proteases exceeds that of intact nuclei 7-fold. The optimum activity of nuclear matrix proteases is observed at pH 8-9. The protease activity of the nuclear matrix is inhibited by p-chloromercuribenzoate, N-ethylmaleimide, EDTA, phenylmethylsulfonyl fluoride. This suggests that thiol, serine and metalloproteases are associated with the nuclear matrix.

Involvement of proteases in apoptosis.

Genetically programmed (apoptotic) cell death plays a key role in cell and tissue homeostasis and in pathogenesis of various diseases. However, the mechanisms involved in apoptotic cell death are poorly understood. At present, the role of proteases in key events of apoptosis is intensively studied and discussed and the involvement of various proteolytic enzymes in the induction and development of the cell death is well-recognized. Proteases of various classes participating in apoptosis have been identified as well as some substrates of these proteases whose cleavage is critical to cell viability; specific protease inhibitors which prevent the cell death have been synthesized. This review summarizes new data on proteolytic enzymes involved in apoptosis and considers the mechanisms of activation of proteases upon induction of apoptosis and the pathways of their involvement in the cell death. The participation of nuclear proteolytic enzymes in the destabilization of chromatin structure and regulation of DNA fragmentation by endonucleases in apoptotic cells is discussed.

[DNA-protein complexes of the nuclear matrix of Ehrlich ascites carcinoma cells]. / DNK-belkovye kompleksy iadernogo matriksa kletok astsitnoi kartsinomy Erlikha.

A DNA-protein complex resistant to 8 M urea and 0.1% SDS was obtained by chromatography of nuclear matrix lysate from Ehrlich ascite carcinoma cells on Sepharose 2BCL. Separation of the complex under more severe conditions (4 M guanidine hydrochloride, 5 M urea) on hydroxylapatite resulted in protein and DNA fractions, as well as in two fractions of the DNA-protein complex. One of the fractions of this complex was enriched with single-stranded DNA and contained a 5-fold excess of newly synthesized DNA over the DNA present in the original complex. The fractions isolated from the nuclear matrix of control Ehrlich ascite carcinoma cells and the cells incubated with novobiocin revealed quantitative and qualitative differences in the electrophoretic patterns of the proteins. Treatment of cells with novobiocin resulted in inhibition of DNA replicative synthesis and an increase in the protein/DNA ratio in the nuclear matrix.

[Action of low-molecular compounds of a polyphenol-quinoid nature from an irradiated organism on cytochrome C]. / O deistvii nizkomolekuliarnykh soedinenii polifenol'no-khinoidnoi prirody iz obluchennogo organizma na tsitokhrom C.

It was shown that gamma-radiation-induced oxidation of phenol compounds in the animal liver, which leads to o-dioxyphenols and quinones accretion, is one of the reasons for excluding cytochrome c from the respiratory chain of mitochondria. Radiochemical oxidation of tyrosil groups at the protein site of cytochrome c and, as a result, the restriction in the rate of reduction of the latter were noted. On the basis of the data obtained the authors discuss the mechanism of weakening the coupling of oxidation to phosphorylation upon irradiation and the molecular mechanism of the so-called cytochrome effect.