Posthypoxic changes in the cerebral cortex of dogs in the late recovery period after 4-hour hypovolemic hypotension]

On the 14th-21st day of the restorative period after four-hour hypovolemic hypotension the level of total RNA decreased in the tissue of the gray matter of the brain by 20.9%, and of DNA-by 13%. In the postmitochondrial supernatant the concentration of prealbumins was reduced by 26.5%, alpha-globulins--19.2%, gamma-globulins--by 59.8%; the concentration of albumins and beta-globulins was increased by 12.6% and 50.0%, respectively. The activity of acid cathepsins rose by 50%, and of acid phosphatase--by 44%. The activity of total lactic dehydrogenase (LDH) and glutamic dehydrogenase failed to differ essentially from the control level. However, LDH isoenzyme spectrum changes towards the reduction of LDH3+4+5 from 31.9 to 14.2%. Analysis of densitograms of electrophoresis in polyacrylamide gel showed physico-chemical changes in the protein molecules similar in nature to the denaturation phenomenon. The Purkinje's cell count decreased in the cerebellum by 41.3% in comparison with control.     

Functional relationship between bovine brain phosphodiesterase activator protein and bovine heart troponin C

Phosphodiesterase activator protein has been purified from bovine brain and its properties compared with that of bovine heart troponin C. While both proteins activate ‘activator depleted phosphodiesterase’ in the presence of Ca²⁺, a 200-fold greater concentration of troponin C was necessary and the maximal effect was less than that with the activator protein. The activator protein formed a Ca²⁺ -dependent complex with bovine heart troponin I during electrophoresis in 6 M urea-polyacrylamide gel. However, the mobility of this complex was different from that of troponin C · troponin I complex and the affinity between troponin C and troponin I was much stronger than that between the activator protein and troponin I. Ca²⁺ induced changes in the electrophoretic mobility of activator protein and the pattern of its elution during gel filtration which were similar to the Ca²⁺-dependent conformational changes observed with troponin C. Bovine heart troponin I reduced basal, troponin C and the activator protein stimulation of phosphodiesterase activity. These results are compatible with the concept that phosphodiesterase activator protein and troponin C might have a functional relationship.     

BRAIN PROTEINS: QUALITATIVE AND QUANTITATIVE CHANGES, SYNTHESIS AND DEGRADATION DURING FETAL DEVELOPMENT OF THE RABBIT

The composition and metabolism of the proteins of the cerebral pallium of the rabbit during the final one-third of the gestational period were measured. During this period, the brain increased in size almost 10-fold and the migration of neuroblasts to form the cerebral cortex became complete. Concurrent with the marked structural changes, the solubility characteristics and electrophoretic distribution of various brain proteins showed little change. However, at the time of birth and in the adult, significant differences in gel electrophoresis patterns were apparent. The rate of synthesis of protein in brain slices from the fetus of 20 days gestation was 3-fold higher per mg of tissue than in the neonate and about 30-fold higher than in the adult. Activities of acidic and neutral proteases per unit weight were virtually the same and nearly constant throughout the late fetal period. However, during this stage, while rapid growth persists, the total protein synthetic activity of the pallium predominated over the total proteolytic activity, whereas sometime after birth the ratios of these activities reversed consequent to a shutdown of the synthetic process.     

A virus-inactivating protein isolated from the digestive juice of the silkworm, Bombyx mori

A protein that can precipitate nuclear polyhedrosis virus (NPV) in vitro was isolated from the digestive juice of silkworm larvae (Bombyx mori) by the procedures of gel filtration and ion-exchange and hydroxylapatite column chromatography. The SDS-polyacrylamide gel electrophoretic and the ultracentrifugal analyses showed that the purified substance was a homogenous simple protein. The molecular weight of the purified protein was 27,000–28,000 and the sedimentation coefficient was 2.61 S. This protein had an additional activity to inactivate NPV of B. mori in vitro, somewhat analogous to serological neutralization by serum proteins. Electron microscope observations showed that amorphous materials could be found on the surface of envelopes and that the nucleocapsids disappeared.     

UV-induced changes of structural and functional properties of blood lactate dehydrogenase isoenzymes in free state and in the presence of serotonin

Photoinduced changes of structural and functional properties of lactatedehydrogenase isoenzymes from human erythrocytes in free state and in the presence of serotonin have been studied by means of gel chromatography, electrophoresis, IR-spectrophotometry and by the method of definition of catalytic activity. UV-light influence induces photoinactivation of erythrocyte's LDH, while its inhibitory effect intensifies with the increase of irradiation dose. The complicated character of changes in electrophoretic mobility and percentage content of isoenzymes LDH-1, LDH-2, LDH-3 under the influence of UV-rays testifies that the decrease of total enzyme activity of these isoforms in connected with their different photosensitiveness and represents the result of many-staged process which is characterized both by the consistent and parallel proceeding of its individual photochemical reactions. A pronounced photoprotective effect of serotonin towards the molecules of erythrocytic LDH isoenzymes has been discovered. It seems to be caused by formation of enzyme--biogenous amine complex affecting the secondary protein structure.     

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.     

Identification of calcium binding proteins in two-dimensional gel electrophoretic pattern of Drosophila thorax and their distribution in two types of muscles

Ten Drosophila thorax proteins (six myosin light chains and four proteins called a, b, c, d) were found to have high affinities with Ca2+. This was proved after subjecting the total Drosophila thorax proteins to two-dimensional (2D) transblot, followed by 45Ca2+ autoradiography. Three proteins (a, c, d) showed Ca2+ dependent electrophoretic mobility changes. To know their tissue-specific localization, fibrillar and tubular type muscle fibers were individually dissected from freeze-dried flies and separately subjected to 2D gel electrophoresis. Fibrillar type muscle had protein b and a small amount of protein a. Tubular type muscle had proteins c, d and a very large amount of protein a. Protein d was characterized to be calmodulin.     

Platelet contractile proteins: separation and characterization of the actin and myosin-like components.

Solution of thrombosthenin, the contractile protein complex isolated from pig platelets, have been studied by analytical ultracentrifugation and zone sedimentation in sucrose density gradients. Freshly prepared thrombosthenin in 0.6 M KCl shows a prominent peak in the ultracentrifuge with S degrees 20w about 5.5 and higher molecular weight aggregates (greater than 100S) sedimenting quickly to the bottom of the cell. Short term storage of high ionic strength solutions of thrombosthenin induces actomyosin-like gel formation and these gels dissociate with ATP and Mg2+ ions into two components of S degrees 20w 8.0 and S degrees 20w50. The supernatant, after actomyosin gel removal, contains only the S degrees 20w5.5 protein. From results of Ca2+ ATPase activity measurements and SDS polyacrylamide gel electrophoretic mobilities of dissociated thrombosthenin separated into fractions in sucrose density gradients, it is concluded that the S degrees20w5.5 protein species is the myosin-like protein of thrombosthenin. The S degrees 20w8.0 protein is not fibrinogen but also has myosin-like properties and is believed to be myosin dimer. Species of higher S values seen in the presence of ATP and Mg2+ in the analytical ultracentrifuge and located in the higher density zones of the sucrose gradients all gave in SDS polyacrylamide gel electrophoresis a single band of molecular weight 46-47,000 daltons. These subunit proteins appear to be derived from a range of polymeric variants of the F-actin-like protein of the contractile complex. All these higher density F-actin-like proteins readily form superprecipitates and display syneresis when combined with rabbit skeletal muscle myosin or platelet myosin. They are also all capable of conferring upon these two myosins a Mg2+ activated ATPase activity. It is suggested that in thrombosthenin solutions a myosin monomer-dimer equilibrium state exists which can be directionally influenced by a number of factors. The coexistence in the solution of F-actin and Mg2+ ATP, for example, increases the propensity of the myosin-like protein to form the higher molecular weight aggregate. Such aggregation may be the initiating mechanism for the intracellular organization of the thick filaments of the actomyosin complex, preparatory to a contractile event.     

Protective effects of antioxidants on age-related changes in the electrophoretic patterns of cardiac LDH,hepatic ALP and serum proteins in male golden hamster

Basal and antioxidant-induced changes in the isoenzyme and isoform patterns of cardiac lactate dehydrogenase (EC 1.1.1.27) and hepatic alkaline phosphatase (EC 3.1.3.1), respectively, as well as the electrophoretic patterns of serum proteins in different age groups of male golden hamster were compared. This is to test whether age-induced changes could be corrected by long-term administration of antioxidants. Data indicated that aging causes no remarkable change in the total activity of either cardiac LDH or hepatic ALP, however a significant increase in the fractional activity of some cardiac LDH isoenzymes and a significant reduction in the fractional activity of some hepatic ALP isoforms were induced by aging. On the other hand, long-term administration of antioxidants appeared to manifest a clear counteracting effect on the age-related changes in old age. This effect was indicated in the fractional activity of cardiac LDH isoenzymes and of hepatic ALP isoforms. The present study has also shown a wide-range variation in serum protein patterns due to aging and/or antioxidant administration, which indirectly reflect a parallel variation in the process of gene expression and/or proteolytic activity.     

Isolation and characterization of calmodulin from an insulin-secreting tumour.

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.     

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the paternal genes for lactate dehydrogenase, glutamate dehydrogenase and acetylcholinesterase in the development of hybrid fish between species from the families of Cobitidae and Cyprinidae]

The time of expression of the paternal genes of glutamate dehydrogenase (GDH), lactate dehydrogenase (LDH) and acetylcholine esterase (AChE) was investigated in the development of fish hybrids. The species which differed by the thermostability of homologous enzymes were selected as parental pairs. The appearance of differences in the thermostability of homologous enzymes between the hybrids and the maternal species suggested the beginning of paternal enzyme synthesis in the hybrid embryos. Differences in the AChE thermostability appeared simultaneously with the enzyme activity at the stage of first muscle contractions (35 hrs of development), differences in the mitochondrial GDH thermostability appeared at the stage of hatching (50-60 hrs) and those in the LDH thermostability 12-17 days after hatching. The total activity of AChE and GDH sharply increased during the period of the paternal enzyme appearance whereas the activity of LDH suffered practically no changes. Differences in the AChE thermostability between the hybrids and the maternal species are the same for both the total AChE (in supernatant, 15,000 gX10 min.) and the solubilised AChE (in supernatant, 130,000 gX60 min.). AChE of the parental species and the hybrids have the same electrophoretic mobility. The differences in the thermostability of enzymes are preserved following the electrophoresis in polyacrilamide gel.     

Changes in nuclear proteins of rat testis cells separated by velocity sedimentation.

The technique of velocity sedimentation at unit gravity has been used to separate rat testis cell suspensions into fractions enriched in particular cell types. Changes in the nuclear proteins from the various fractions have been characterized by polyacrylamide gel electrophoresis, and correlated with the changing morphology of the nucleus during spermatogenesis. The most striking alterations in both protein composition and nuclear morphology occur during spermatid maturation as both histone and non-histone proteins are replaced by highly basic, low molecular weight, spermatidal proteins. This replacement process is accompanied by a quantitative reduction in both histone and non-histone proteins. The synthesis of at least three basic proteins has been identified with late stage spermatids. One of these proteins is a highly basic sperm-specific protein containing high levels of cyst(e)ine and arginine. A second protein synthesized in late stage spermatids is lysine rich, while the third protein contains cyst(e)ine and co-migrates with histone F2a1 on acid-urea polyacrylamide gels. The changes in protein composition of rat testis nuclei after irradiation or hypophysectomy reflect the resulting changes in the cellular composition of the testis. After selective elimination of the germinal cells by irradiation, the electrophoretic pattern of acid-soluble proteins from the testis is very similar to that of somatic tissue. Thus, the cellular specificity of nuclear proteins demonstrated here using cell separation techniques is also apparent following treatments which selectively alter the cellular composition of the testis.     

Proteins of soybean seeds: I. Isolation and characterization of the major components

Soybean (Glycine max) storage proteins were characterized by sedimentation and by polyacrylamide gel electrophoresis under dissociating (8 m urea) and nondissociating conditions. Three sedimenting classes of proteins were found, with sedimentation coefficients of 2.2S, 7.5S, and 11.8S. The coefficients were related to the bands obtained by electrophoretic separation. The results support the idea that relatively few proteins make up the bulk of the seed protein.     

Compositional changes in soluble proteins of cerebral mantle,cerebellum, and brain stem of rat brain during development

Soluble proteins from the cerebral mantle, cerebellum, and brain stem of rat brains were analyzed at various developmental stages by a two-dimensional gel electrophoresis technique. The electrophoretic technique resolved the soluble proteins into 100–150 polypeptide spots on two-dimensional gels and gave reproducible and highly resolved profiles of them. Although most of major polypeptides were commonly found in all the three brain regions, some polypeptides were shown to be unique to a specific brain region. Each brain region was different in the electrophoretic profile of soluble proteins at every developmental stage examined, although there was considerable similarity in the profiles of each of the three brain regions in fetal animals (16–17 days), indicating that soluble proteins undergo different compositional changes in each of the three brain regions during postnatal development. In addition, the number of polypeptide spots on the electrophoretic profile increased remarkably during postnatal development in all of the three brain regions, suggesting that soluble proteins become more heterogeneous during postnatal development in each of the three brain regions.     

A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization

Rat brain tryptophan 5-monooxygenase was activated by incubation with ATP, Mg2+, calmodulin, and micromolar concentrations of Ca2+. The activating activity was resolved into two distinct peaks upon gel filtration on Sepharose CL-6B: one, Ca2+-, calmodulin-dependent protein kinase, and the other, a heat-labile activator protein. The activator protein was purified to apparent homogeneity from rat brain by a procedure involving calmodulin-Sepharose 4B, Sephadex G-150, and phenyl-Sepharose CL-4B column chromatography. The molecular weight of the activator protein was determined to be 70,000 by sedimentation equilibrium and by gel filtration on Sephadex G-150. The protein gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 35,000, indicating that the protein might be composed of two identical subunits. Analysis of cross-linked activator protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also suggested that the protein might be a dimer of identical subunits. Some other molecular properties of the activator protein were: sedimentation coefficient, 4.3 S; Stokes radius, 3.6 nm; diffusion coefficient, 6.0 x 10(-7) cm2/s; frictional ratio, 1.32; and partial specific volume, 0.73 cm3/g. The activator protein activated tyrosine 5-monooxygenase as well as tryptophan 5-monooxygenase in the presence of ATP, Mg2+, Ca2+, calmodulin, and Ca2+-, calmodulin-dependent protein kinase.     

A method for quantitative determinations of nonhistone proteins in chromatin.

An electrophoretic method on cellogel strips at alkaline pH is used to determine the amount of nonhistone proteins in chromatins in a very reproducible way after isolation of the total chromatin proteins in 2m NaCl or 2m CsCl + 5m urea. The procedure is demonstrated with calf thymus, rat liver, and pig brain chromatin. The influence of the chromatin preparation on the protein composition is discussed.     

Adenylate cyclase activity and cyclic AMP content in brain tissue of dogs during clinical death and in the post-resuscitation period

The cAMP levels and adenylate cyclase activity have been studied in the grey brain substance and striatum system of dogs during circulation arrest due to electrotrauma of different duration (1-2, 15, 45 min) and in postresuscitation period in animals recovered after 15-min clinical death. Adenylate cyclase is strongly activated and cAMP levels are increased in the brain areas under study during complete brain ischemia. The cAMP levels in the grey substance and in striatum system are reduced considerably compared to the control, accounting for 12-20 on days 2-5 of postresuscitation period in animals with neurologic deficit. Adenylate cyclase and phosphodiesterase enzyme activity is twice higher in the striatum system and 50% lower in the grey brain substance than the baseline. The disturbances in cyclic nucleotide exchange, along with other factors seem to play an important role in the pathogenesis of postresuscitation encephalopathy.     

High levels of a calcium-dependent modulator protein in spermatozoa and its similarity to brain modulator protein

Sperm from hamster, human, rooster, rabbit and sea urchin were found to contain relatively high levels of calcium-dependent modulator protein. Using rabbit sperm the modulator protein was found to be exclusively located in the sperm head fraction (nuclei + acrosomes) with no activity present in the midpiece or tail regions. The modulator protein represents approximately 12% of the total soluble protein found in the sperm head fraction and is similar to porcine and brain modulator proteins in its ability to activate brain cyclic nucleotide phosphodiesterase, its heat stability and electrophoretic migration. We have also observed modulator protein to be present in high levels in sea urchin eggs.     

Contractile proteins in the fractions of soluble and insoluble chromatin of liver and thymus

The proteins corresponding in molecular weight and solubility in salt solutions to skeletal muscle actin and myosin were revealed in liver and thymus chromatin fragments. When the ionic strength reached 0.3, about 60% of the myosin-like protein identified by electrophoretic mobility of high chains and the K+-EDTA-ATPase activity was cosedimented with nucleohistones. In the presence of ATP or PPi and Mg2+ the solubility of myosin in such salt solutions increased up to 90%, which was paralleled with significant stimulation of RNA release from the nucleohistones. The conformity in the degree of extraction and sedimentation of RNA and intranuclear myosin was also observed in other solutions used during myosin purification. The supposition that the nuclear system of contractile proteins causes labile, ATP-dependent binding of RNA to chromatin is discussed. No essential differences in the actin or myosin contents in the fractions of soluble and non-soluble chromatin were detected.