Association of proteinases with histones from rat thymus nuclei]

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.     

Differential elution of histones from gel-trapped nuclei

A method has been developed for characterizing at, submicrogram levels the heterogeneity of histones from purified nuclei. The histones are eluted with a smooth concentration gradient from nuclei trapped in polyacrylamide–gel threads and are collected in a micro fraction collector suitable for volumes in the 10–100 μl range. The gradient and fraction collection systems are governed by cam driven syringes. Samples obtained are subjected to electrophoresis in a starch-gel system and the gels are stained with a highly sensitive stain specific for guanidinium groups. Seven major and a similar number of minor components are demonstrated in the histones. The method of differential elution of trapped macromolecules is suitable for use with systems other than nuclei and histones.     

Proteinase activity of the peripheral blood cell nuclei of gamma-irradiated rats

Immediately after gamma-irradiation of rats a dose-dependent increase in the activity of alkaline and Ca-dependent proteinases was exhibited by leukocyte nuclei. In nuclei of granulocytes, in contrast to those of lymphocytes, radiation activation of proteinases was more pronounced.     

Metabolism of histones and non-histone proteins in liver cell nuclei and chromatin from rats of various ages

The metabolism of various classes of histones and nonhistone proteins in intact nuclei and in liver chromatin of albino Wistar rats aged 1, 3, 12 and 24 months, was studied. It was shown that in the course of postnatal development the metabolism of nonhistone proteins extracted with 0.14 M NaCl in murine liver is increased. Later in ontogenesis, the incorporation of labeled precursors into proteins HMG 14 and HMG 17 decreases; the specific radioactivity of proteins HMG 1 + 2 is higher in 3- and 24-month-old animals. The intensity of metabolism of nonhistone proteins and histones is higher within the composition of the chromatin complex than in the intact nucleus at all stages of postnatal development. Among other histone proteins, histones H1 are characterized by the highest level specific radioactivity in rats of all age groups.     

A set of non-histone proteins isolated from the nuclei of various rat tissues

A set of non-histone proteins has been identified in the nuclei from liver, brain, spleen and testis tissues of the rat. Following moderate digestion of thoroughly washed nuclei with DNase I or micrococcal nuclease, EDTA was added to 5 mM to the reaction mixture and the preparation centrifuged. We found that the supernatant contained a limited amount of non-histone proteins (fraction S1). Sodium dodecyl sulfate (SDS) gel electrophoresis revealed S1 to be composed of a remarkably simple set of proteins resolved into four groups (A-D) each possessing closely spaced doublets or a triplet. Their molecular weights were A, 76 100-80 000; B, 48 200-49 500; C, 44 500-45 200 and D, 39 500-41 500. The yield suggested that these proteins were structural constituents; however, they did not coincide with the known structural proteins of the cell nucleus. Two-dimensional gel electrophoresis further resolved each of the SDS bands into as many as nine spots, according to various charges. Some were labelled with [32P]orthophosphate in vivo, or with [gamma-32P]ATP and purified nuclear protein kinase NII in vitro. The released proteins B-D had fairly constant relative molar ratios at various times of digestion, thereby indicating possible localizations at similar sites in the nucleus. The kinetic data together with the aggregation property at neutral pH values and the solubility in 5 mM EDTA suggest that proteins B-D constitute a group of proteins that have several common characteristics.     

ADP-ribosylation of proteins in nuclei and mitochondria from tissues rats of various age exposed gamma-radiation

Constitutive and gamma-induced ADP-ribosylation of nuclei and mitochondrial proteins in 2- and 29-month-old rats was studied. ADP-ribosylation was determined by binding of [3H]-adenin with the proteins after incubation of cellular organells in reaction mixture supplemented with [adenin-2,8-3H]-NAD. It was detected that the level of total protein ADP-ribosylation in the nuclei is 4.5-6.2 times higher than in the mitochondria. By inhibition of poly(ADP-ribose) polymerase (PARP) with 3-aminobenzamidine and treatment of ADP-ribosylated proteins with phosphodiesterase I, it was demonstrated that about 90% of [3H]-adenin bound by proteins in the nuclei and 70% in the mitochondria was the result of PARP activity. The level of total ADP-ribosylation of nuclear and mitochondrial proteins in the tissues of old rats was reliably lower than in young animals. This reduction of ADP-ribosylation in old animals is the result of the lower activity of PARP, not of mono(ADP-ribosyl) transferase (MART). The level of ADP-ribosylation of proteins in the nuclei of brain and spleen cells of 2-month-old rats irradiated with of 5 and 10 Gy was by 49-109% higher than in the control. At the same doses of radiation, the level of ADP-ribosylation of nuclear proteins in brain and spleen of old rats increased only by 29-65% compared to the control. Unlike cell nuclei, the radiation-induced activation of ADP-ribosylation in mitochondria was less expressed: the level of ADP-ribosylation increased by 34-37% in young rats and by 11-27% in old animals. This increased binding of ADP-ribose residues by the proteins of nuclei and mitochondria from tissues of gamma-irradiated rats is exceptionally conditioned by activation of poly(ADP-ribosyl)ation because the level of mono(ADP-ribosyl)ation remains constant. The results of this study enable the suggestion that poly(ADP-ribosyl)ation also occurs in the mitochondria of brain and spleen cells of the gamma-irradiated rats, though less pronounced than in cell the cell nuclei of these tissues. Thus, one of the probable causes of the less efficient repair of radiation-induced DNA damage in old organisms is a decline of both constitutive and induced poly(ADP-ribosyl)ation of proteins in cell nucleus and mitochondria.     

Characterization of histones from beef pancreas]

The total histone of ox pancreas was fractionated by electrophoresis on 10 and 25 cm polyacrylamide gels according to Panyim and Chalkley (1969). The presence of an additional subfraction within the lysine rich histone was stated. In the course of the fractionation of total histone according to the method of Oliver et al. (1972) this additional histone component was extracted together with F1 histone.     

Potential mechanisms of effects of endogenous neuromodulators on the interdependent activity of neurons in various nuclei of the basal ganglia

A possible mechanism of influence of neuromodulators on interdependent activity of neurons in the diverse basal ganglia nuclei is suggested. According to modulation rules, an activation of postsynaptic Gs- or Gq/11-(Gi/0-) protein coupled receptors promotes induction of long-term potentiation (depression) of excitatory inputs to different neurons and augmentation (lowering) of their activity; an activation of presynaptic Gs- or Gq/11-(Gi/0-) protein coupled receptors promotes a rise (decrease) of release of GABA and co-peptides from striatal terminals and glutamate release from subthalamic terminals in the globus pallidus and output nuclei. It follows from the modulation rules that, since identical receptors are present on striatal neuron and their axon terminals, effects of neuromodulator action in diverse basal ganglia nuclei can be summarized. Neuromodulators released from striato-nigral and striato-pallidal fibers could promote interdependent activity of neurons in "direct" and "indirect" pathways through the basal ganglia due to convergence of these fibers on cholinergic interneurons and pallido-striatal cells.