Activity and properties of cycl AMP-dependent protein kinases in the gray substance of the rat brain in acute radiation injury

It was shown that in conditions of acute radiation affection there was a decrease of 37% and 32% in activity of cyclic AMP-dependent protein kinases, isolated from cytosol of gray substance of rat brain cells and from the synaptic membranes solubilized by triton X-100, respectively. The activity of enzymes from the control and irradiated animals was maximum with similar concentration of 3':5'-AMP (10(-6) M) and pH (7.4) in Tris-HCl and HEPES-buffers. Histone H4 was shown to be the best substrate for both enzyme forms. Other substrates under study were phosphorylated to a lesser degree.     

Molecular forms of acid proteinases of the brain]

Molecular forms of cathepsin D bound with subcellular structures were studied in the grey matter of the large hemispheres. Free and bound forms of the enzymes exposed to solubilization with detergent triton X-100 were fractionated by passage through a Sephadex G-100 column. Gel chromatographic analysis demonstrated three peaks of acid proteinase activity. Different areas of solubilization curves of acid proteinases corresponded to different molecular forms of cathepsins. The initial S-shape areas of solubilization curve corresponded to the first high molecular weight peak of the enzyme activity in the grey matter, whereas the subsequent linear ones -- to the second peak; the activity of free forms of the enzyme corresponded to the third peak.     

五种鼠的脑和头骨形态及其生态关系

通过测量棕色田鼠（Microtus mandarinus）、沼泽田鼠（Microutus fostis）、甘肃鼢鼠（Myospalax can-sus）、大仓鼠（Cricelulus triton）、黑线姬鼠（Apodomus agraius）的头骨和脑外部形态,计算其长度与体长的比值、脑重与体重的比值及其发头化指数。发现棕色田鼠和甘肃鼢鼠的宽与体长之比、齿列长与体长之比显著大于大仓鼠和黑线姬鼠,这些特征和各自的食物类型紧密相关。棕色田鼠脑颅测量值和体长之比、脑重和体重之比较大,甘肃鼢鼠以上值较小,发头化指数由大到小依次为：棕色田鼠、沼泽田鼠、黑线姬鼠、大仓鼠、甘肃鼢鼠、小白鼠、大白鼠。棕白田鼠脑除嗅球外无性二型,而且大脑测量值相对较大。以上脑特征可能和棕色田鼠单配,制婚配制度有关。而甘肃鼢鼠大脑较小的原因可能和其多配制婚配制及其独居生活有关。甘肃鼢鼠和棕色田鼠的小脑测量值与体长之比小于沼泽田鼠、大仓鼠、黑线姬鼠;甘肃鼢鼠的视神经很细,棕色田鼠视神经较细,而其他3种鼠视神经都较粗;甘肃鼢鼠的嗅球较大;这些特征与它们分别适应于地下和地上生活方式有关。     

ONTOGENETIC DEVELOPMENT OF A PHOSPHODIESTERASE ACTIVATOR AND THE MULTIPLE FORMS OF CYCLIC AMP PHOSPHODIESTERASE OF RAT BRAIN

Abstract— The activity of cyclic AMP phosphodiesterase of rat cerebral homogenates increased several-fold between 1 and 60 days of age. Enzyme activity in the cerebellum, on the other hand, did not increase during this period. A kinetic analysis of the phosphodiesterase activity revealed evidence for multiple forms of the enzyme and indicated that the postnatal increase in phosphodiesterase activity of rat cerebrum was due almost exclusively to the high K_m enzyme. In cerebellum, the ratio of the high and low K_m enzyme remained fairly constant during ontogenetic development. Physical separation of the phosphodiesterases contained in 100,000 g soluble supernatant fractions of sonicated brain homogenates by polyacrylamide disc gel electrophoresis confirmed the presence of multiple enzyme forms. In adult rats we found six distinct peaks of phosphodiesterase activity (designated I to VI according to the order in which they were eluted from the column) in cerebellum and 4 forms of the enzyme (Peaks I through IV) in cerebrum. Brains of newborn rats had a different pattern and ratio of phosphodiesterase activities. For example, Peak I phosphodiesterase was undetectable in cerebrum or cerebellum of newborn rats. Moreover, in the cerebellum of newborn rats Peak II was the dominant peak whereas in the cerebellum of adult rats Peak III was the largest peak. A comparison of the multiple forms of phosphodiesterase from the cerebrum of newborn and adult animals suggested that the postnatal increase in phosphodiesterase activity previously seen in crude homogenates was due largely to an increase in a high K, Peak II phosphodiesterase. The ratios of activities of the other peaks and their sensitivities to an activator of phosphodiesterase were similar in newborn and adult rats. An endogenous heat-stable activator of phosphodiesterase was found in cerebrum, cerebellum and brain stem. In newborn rats, the cerebellum contained several-fold less activity of this activator than did cerebrum or brain stem. However, the activity of this activator increased with age in the cerebellum and would appear to have decreased postnatally in cerebrum and brain stem. These results suggest that some multiple forms of phosphodiesterase can develop independently and that changes in activities of these phosphodiesterases may occur by increases in the quantity of enzyme or by changes in the quantity of an endogenous activator of phosphodiesterase.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

Association of prolyl hydroxylase activity with membranes

Addition of ionic and nonionic detergents to whole homogenates of liver, kidney and lung prepared by a mild homogenization technique resulted in a two- to three-fold increase of prolyl hydroxylase activity. After subcellular fractionation of whole homogenates of liver, particulate and supernatant fractions were incubated in the presence and absence of triton X-100 and assayed for prolyl hydroxylase activity. All particulate fractions tested were able to release significant amounts of prolyl hydroxylase activity in the presence of triton. The release of enzyme activity by triton was observed with the 1000 × g and 17,000 × g supernatants but not with the 105,000 × g supernatant; thus indicating that detergent does not activate soluble enzyme nor make the substrate more accessible to hydroxylation by the enzyme during incubation. Rigorous homogenization of the 17,000 × g particulate fraction with the Polytron ST system resulted in a substantial loss of the amount of prolyl hydroxylase activity released by treatment with triton. These data suggest that a significant amount of prolyl hydroxylase activity is associated with membranes under physiological conditions.     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

Isolation and properties of trypsin inhibitor from the leaves of pedunculate oak]

Four protein fractions inhibiting trypsin are isolated from the English oak leaves by the method of chromatography on DEAE-cellulose. Three active fractions more are found in each of them after the affinity chromatography on trypsin-agarose. Each of 12 multiple forms in the calcium-free medium contains different sets of proteins and oligopeptides possessing rather high inhibiting activity. Ca2+ being introduced to the medium, all the multiple forms somewhat increase their activity, having one peak (Mm approximately 16.5 kDa) on the column with sephadex G-50. The inhibitor possesses high indices of denaturation stability and specificity to trypsin.     

The heterogeneity of sorbitol dehydrogenase from the cytoplasm of mammalian brain cells

The multiple molecular forms of sorbitoldehydrogenase in cytoplasm of brain cells of bull, ground squirrel, guinea-pig, rat, hamster and mouse have been found using the method of electrophoresis in polyacrylamide gel and the subsequent specific dyeing for the fermentative activity. All revealed zones of activity are related to the slowly migrating ones. A set of multiple molecular forms from different sources is various. A form with relative electrophoretic activity 0.385 is found in all analyzed animals. The conditions for obtaining of distinct zones of activity on zymograms are chosen.     

Multiple forms of endothelial cell growth factor. Rapid isolation and biological and chemical characterization

Endothelial cell growth factor (ECGF) can be rapidly purified from bovine brain to high specific activity using heparin-Sepharose affinity chromatography. Purification of the mitogen by this method results in relatively high yields of the polypeptide (10 to 100 micrograms/kg of tissue) with biological activity on murine and human endothelial cells in the picogram range. The product obtained is a mixture of two single-chain polypeptides with apparent molecular weights of 17,000 (alpha-ECGF) and 20,000 (beta-ECGF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two forms of ECGF can be separated by either NaCl gradient elution from heparin-Sepharose or reversed-phase high pressure liquid chromatography. The two polypeptides are related on the basis of similar: amino acid compositions, affinity for heparin-Sepharose, cyanogen bromide and trypsin-derived cleavage products, and biological activity. Furthermore, the cyanogen bromide fragments derived from the two forms of ECGF also possess similar amino acid compositions and mobilities on sodium dodecyl sulfate gels. These data suggest that there are at least two discrete molecular forms of ECGF in bovine brain and that these two molecules are structurally related.     

Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species

The relative efficiency of a buffered medium containing a high salt concentration and EDTA as a means to solubilize collagen-tailed molecular forms of acetylcholinesterase has been examined in four brain areas of several species belonging to different vertebrate classes. This extraction procedure has proved successful in most cases, with the yield of tailed enzyme varying between less than 1 and 26% of the total tissue activity. The solubilization values are consistently higher in more primitive vertebrates than in mammals and, for a given species, are usually lower in the telencephalon than in other brain structures. Our results confirm that the vertebrate central nervous system contains collagen-tailed quaternary structural forms of acetylcholinesterase.     

Localization of Membrane-Bound Catechol-O-Methyltransferase

Abstract: Previous studies of the distribution of catechol-O-methyltransferase (COMT) have concentrated on the soluble enzyme activity. In this study the activity of the membrane-bound enzyme was determined in different brain regions and peripheral tissues of the rat. Membrane-bound COMT, like the soluble enzyme, has a general distribution with high levels in liver, kidney, and vas deferens. However, the ratio of membrane-bound to soluble activity varies almost 30-fold in different tissues, with the highest ratio in brain. Membrane-bound activity varies twofold within brain. In view of their different localization and kinetic properties, it seems likely that the two forms of COMT have different functions in vivo.     

Changes in the protein composition of the synaptic structures of the brain of rats in tetanus intoxication]

As shown by the method of electrophoresis on polyacrylamide gel, the number of proteins with low electrophoretic mobility proved to be increased in the triton extract fractions of synaptic structures isolated from spinal cord of rats with local tetanus; no changes in the protein spectrum were revealed in the dodecyl-sulphate extract. In vitro tetanus toxin stimulated the lysin-H3 incorporation into the total proteins of synaptosomes of rat brain cortex.     

High aryl acylamidase activity associated with cobra venom acetylcholinesterase: Biological significance

Investigation of the non-classical functions of cholinesterases (ChEs) has been the subject of interest in the past three decades. One of which is aryl acylamidase (AAA) activity associated with ChEs, but characterized in in vitro, as an enzyme, splitting the artificial substrate o-nitroacetanilide with unknown physiological function. In the present study, we have compared levels of AAA activity of AChE from different sources like goat brain, electric eel organ and from venoms of different snakes. Remarkably cobra venom showed the highest AAA activity and also high AAA/AChE ratio. Both serotonergenic and cholinergic inhibitors inhibited the cobra venom AAA activity in a concentration dependent manner, which also underlines the association of AAA with AChE of cobra venom. The study becomes interesting because of i) the cobra venom AChE exists in monomeric globular forms; ii) in Alzheimer's disease too the most abundant forms of cholinesterases are monomeric globular forms, thought to be involved in the pathogenesis of Alzheimer's disease; iii) the effect of Alzheimer's disease drugs on the AAA activity of cobra venom, indicated that AAA activity of cobra venom was more sensitive than AChE and iv) Huperzine and Tacrine showed more pronounced effect on AAA. Thus, this study elucidates the high AAA associated with cobra venom AChE may serve as one of the prominent activity to test the pharmacological effect of AD drugs, as other sources were found to have lower activity.     

Di-Ras2 Protein Forms a Complex with SmgGDS Protein in Brain Cytosol in Order to Be in a Low Affinity State for Guanine Nucleotides

The Ras family of small GTPases function in a wide variety of biological processes as “molecular switches” by cycling between inactive GDP-bound and active GTP-bound forms. Di-Ras1 and Di-Ras2 were originally identified as small GTPases forming a distinct subgroup of the Ras family. Di-Ras1/Di-Ras2 mRNAs are detected predominantly in brain and heart tissues. Biochemical analysis of Di-Ras1/Di-Ras2 has revealed that they have little GTPase activity and that their intrinsic guanine-nucleotide exchange rates are much faster than that of H-Ras. Yet little is known about the biological role(s) of Di-Ras1/Di-Ras2 or of how their activities are regulated. In the present study we found that endogenous Di-Ras2 co-purifies with SmgGDS from rat brain cytosol. Size-exclusion chromatography of purified recombinant proteins showed that Di-Ras2 forms a high affinity complex with SmgGDS. SmgGDS is a guanine nucleotide exchange factor with multiple armadillo repeats and has recently been shown to specifically activate RhoA and RhoC. In contrast to the effect on RhoA, SmgGDS does not act as a guanine nucleotide exchange factor for Di-Ras2 but instead tightly associates with Di-Ras2 to reduce its binding affinity for guanine nucleotides. Finally, pulse-chase analysis revealed that Di-Ras2 binds, in a C-terminal CAAX motif-dependent manner, to SmgGDS immediately after its synthesis. This leads to increased Di-Ras2 stability. We thus propose that isoprenylated Di-Ras2 forms a tight complex with SmgGDS in cytosol immediately after its synthesis, which lowers its affinity for guanine nucleotides.     

Picrotoxinin and Phorbol-12-Myristate-13-Acetate Stimulate the Secretion of Multiple Forms of Somatostatin from Cultured Rat Brain Cells

The molecular forms of somatostatin (SRIF) secreted by cultured fetal rat brain cells were resolved using reverse phase high performance liquid chromatography followed by radioimmunoassay. Multiple forms of SRIF-like immunoactivity were detected in media from cells treated with either picrotoxinin, phorbol-12-myristate-13-acetate, or high potassium. For stimulated cells, elevated levels of an SRIF-28-like molecule, an SRIF-14-like molecule, and a hydrophobic SRIF-like molecule were observed compared to basal conditions. All three forms of SRIF-like molecules were also detected in acid extracts of whole cells. The data are consistent with the possibility that secretion of multiple SRIFs , including SRIF-28, may be regulated by multiple effectors and mechanisms.     

Multiple molecular forms of glia maturation factor.

Glia maturation factor from the pig brain can be detected in two molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated by papain, ficin and pronase but resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation by low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.     

雄性大仓鼠体重对其斗殴行为及社会等级的作用

在实验室条件下测定雄性大仓鼠体重对社会等级和斗殴行为的作用模式,检验体重对雄性大仓鼠社会等级及斗殴行为序列具有重要影响的假设。本实验以16只成年雄性大仓鼠为目标个体,采用等级内部的线性概率、组内循环三元组数量（d）和优势等级的线性度（K）,排列个体的社会等级序位。研究结果表明,雄性大仓鼠可形成近似线性的优势等级,体重与个体的优势等级,攻击行为和胁腺标记行为均呈显著的正相关关系,与防御行为和攻击潜伏期存在显著的负相关关系。说明独居性物种大仓鼠雄体间可形成优势等级关系,体重对此关系具有重要的作用。     

Peptide mapping of multiple forms of cyclic nucleotide phosphodiesterase

Purified multiple forms of 3':5'-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17) were analyzed using two-dimensional tryptic pep]tide mapping of radioiodinated peptides. Comparisons of peptide maps of rat liver insulin-sensitive phosphodiesterase (PDE) with rat brain calmodulin-sensitive PDE suggest that some peptides co-migrate (31-43% co-migration). However, except for a single peptide, bovine retinal rod outer segment PDE, peptide maps appear unrelated to the other two forms (7-12% co-migration). In contrast, peptide maps of a 36,000-dalton proteolysis product of calmodulin-sensitive PDE are highly related to the peptide maps of a rat brain calmodulin-sensitive holoenzyme (73% co-migration). These results suggest that the multiple PDE forms are distinct molecular entities.     

Distributional and developmental variations of multiple forms of calpastatin in mouse brain

DEAE-cellulose chromatography of mouse brain extract demonstrated the occurrence of two calpastatin fractions, CS-0.1 and CS-0.2, with distinctly higher content of the latter. CS-0.1 emerged from the column at 0.1 M NaCl, inhibited calpain II more strongly than calpain I, and identified also immunologically with hitherto known calpastatin. CS-0.2 emerged at 0.2 M NaCl, inhibited calpain I more strongly than calpain II, and did not crossreact with anti-calpastatin antibody used. Fairly consistent amounts of CS-0.2 and calpain II were found in the brain of mice from 10 days to 10 weeks after birth, while CS-0.1 became measurable only after 4-week growth. In adult mice, CS-0.1 was highest in specific activity in brainstem, lower in cerebellum, and not detectable in cerebral hemisphere. Physiological significance of multiple forms of calpastatin and their variations found is not known.