Chromatin protein metabolism and phosphorylation in the cells of different parts of the brain and liver in rats under normal conditions and under motor load

The relationship of intensive motional load with quantitative changes of the synthesis processes and phosphorylation in chromatin peptide fractions of varied polyacrylamide gel electrophoretic mobility from different rat brain structures and liver has been investigated. It has been established that the functional influences change not only the velocity of metabolism and phosphorylation but also the pattern of chromatin protein distribution. The new low molecular peptides differing in their electrophoretical mobility appear in chromatin of liver and neocortical neurons. The changes of the synthesis processes and phosphorylation typical of some fractions of the cerebral chromatin are variable and not so important as in the case of cytoplasmic proteins. The velocity of synthesis of the most proteins studied and the phosphorylation rate of some proteins increase in the neocortical neurones. The phosphorylation rate of separate low molecular peptides increases in the glial cells.     

Incorporation of amino acids into liver and serum proteins during prolonged administration to animals of ethanol and cholesterol

The synthesis of soluble liver proteins and amino acid incorporation into blood serum proteins were studied in guinea pigs which were daily administered ethanol and cholesterol during 3 months. 9 fractions obtained by electrophoresis in polyacrylamide gel were analyzed. It has been found that ethanol and cholesterol in the liver suppress the synthesis of 4 out of 9 recorded protein fractions. In the serum the ethanol effect against a background of the cholesterol administration is characterized by a sharp decrease of specific radioactivity of albumins while the quantity of the protein fraction is unchanged. These results together with the data available in literature on the ethanol suppression of proteolysis suppose that the joint effect of ethanol and cholesterol is accompanied by a decrease in the blood serum albumin decomposition.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

ANALYSE ELECTROPHORETIQUE COMPAREE DES PROTEINES CEREBRALES TOTALES, SOLUBLES ET PARTICULAIRES, CHEZ CINQ SOUCHES DE SOURIS

Abstract—

• 1 The distribution of total cerebral proteins from five strains of adult mice: Quaking (Qk), C57BL/6, CBA, DBA/2 and C57BR have been compared according to various subcellular brain fractions. The saline or detergent-soluble proteins were separated in a pH discontinuous system by analytical disc electrophoresis on polyacrylamide gel, either at pH 8·9 (acidic proteins) or pH 4·3 (basic proteins).
• 2 Significant quantitative differences and higher electrophoretic mobility were observed both in the high molecular weight acidic proteins of myelin and in the low molecular weight soluble proteins of myelinic and synaptosomal fractions of the Quaking mutant. Differences between Qk and control were inapparent in nuclear, mitochondrial or microsomal protein fractions.
• 3 The in vivo incorporation of tritiated amino acids into the brain proteins of the Qk mouse have been studied. An increased level of incorporation was found in two soluble, acidic proteins of the supernatant cell sap.
• 4 The electrophoretic patterns of the brain proteins from three other inbred strains (CBA, DBA/2 and C57BR) were identical except DBA/2, whose soluble and acidic proteins of the supernatant cell sap were characterized by two supplementary minor bands. The observed protein abnormalities have been discussed in relation to the alteration of the CNS myelination and to the genetic lesions presumed to be responsible for a cellular multienzymic induction.

     

Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver tissue sources. Indeed approximately one-third of the proteins identified in the soluble fractions are associated predominantly to one of the three tissues, indicating a tissue-dependent regulation of mitochondrial proteins. Furthermore a small percentage of the mitochondrial proteome is unique to each tissue.     

NUCLEAR PROTEINS OF THE GUINEA PIG BRAIN

Abstract—

• 1 Chromatin protein fractions were separated from the nuclei from brain, liver and kidney of the guinea pig. The fractions were studied by electrophoretic methods and amino acid analysis.
• 2 Brain nuclear fractions were washed with 0.15m -NaCl and nuclear acidic proteins then removed by 0.35 m -NaCl. These 0.35 m -NaCl-extracted proteins were considered to be similar to the nuclear soluble acidic proteins.
• 3 Nonhistone-1, histone and nonhistone-2 fractions were obtained from 2.0 m -NaCl-soluble chromatin fractions by lowering the salt concentration and successive extraction with acid and alkali. The nonhistone-3 fraction was also extracted from the nuclear residue by alkaline solution.
• 4 The contents and characteristics of the nonhistone fractions of the brain, especially the nonhistone-1 fraction, differed among the three tissues. The histone fractions showed no obvious difference among the three tissues. The nonhistone-1 fraction of the brain, which comprised a low percentage of total nuclear protein, contained relatively high amounts of acidic proteins.

     

Use of immunochemical methods for characterization and purification of tissue-specific inhibitors of mitotic activity (chalones)]

The 81% ethanol-precipitable fraction of water-soluble proteins from skin inhibiting the proliferation of epidermal tissues was shown to contain 9 protein components according to acrylamide gel disk-electrophoresis. Seven of these were identical to homologous serum proteins and could be adsorbed on the corresponding immunosorbents. Two proteins remained in the solution after immunoadsorption. Their electrophoretic properties were the same as those in the total 81% ethanol fractions. These proteins like the 81% ethanol fraction inhibited the entering of cells into mitosis and DNA synthesis phase.     

Concentrations of high-mobility-group proteins in the nucleus and cytoplasm of several rat tissues

Nuclear and cytoplasmic fractions were isolated from various tissues of the rat by a nonaqueous technique. The high-mobility-group (HMG) proteins were extracted from these fractions with acid and separated by one- and two-dimensional PAGE. The concentrations of high-mobility-group proteins HMG1, HMG2, and HMG17 in the nucleus and cytoplasm were then estimated from the staining intensities of the electrophoretic bands. The cytoplasmic concentrations of these proteins were very low--usually less than 1/30 of those present in the corresponding nuclear fractions. For the tissues studied (liver, kidney, heart, and lung), the concentrations of HMG proteins in the nucleus did not differ significantly from one tissue to another. Averaged over the four tissues investigated, there were 0.28 molecule of HMG1, 0.18 molecule of HMG2, and 0.46 molecule of HMG17 per nucleosome. These values are considerably higher than those that have been reported previously.     

Protein tyrosine phosphorylation in normal rat tissues

• 1.1. Exogenous and endogenous tyrosine protein phosphorylation activities were examined in soluble and partieulate fractions from various normal tissues by using poly-[Glu-⁸⁰Na, Tyr²⁰] and a monoclonal antibody specific for phosphotyrosine.
• 2.2. Phosphorylation of the exogenous substrate by the partieulate forms of TPKs was 2- to 10-fold higher than by soluble forms. The activities of partieulate and soluble enzymes decreased in the following order: spleen > (thymus = kidney) > testes ⩾ (pancreas = liver = brain) > heart.
• 3.3. The level of endogenous phosphorylation in the tissues decreased respectively in the following order: thymus > brain ⩾ (pancreas = liver) > spleen > testes > kidney > heart for the partieulate fractions, and spleen > thymus > brain > pancreas ⩾ liver > testes > kidney > heart for the soluble fractions.
• 4.4. A large number of phosphotyrosine-containing proteins were detected. In addition, several phosphotyrosine-containing proteins of similar molecular weight were found in different tissues and fractions.

     

Protein Metabolism of Allium Radicle Tips during Germination

As an initial step in the study of the molecular events surrounding the establishment of a mature root apex, analyses of the early germinative growth of the Allium radicle were undertaken. Changes in the level and pattern of the soluble proteins were investigated by polyacrylamide gel disc electrophoresis and Joyce-Loebl densitometry. Histochemical studies revealed the presence of protein bodies in the terminal mm of the Allium radicle. These bodies underwent depletion and breakdown during germination. The results of the electrophoretic fractionation of the soluble proteins during germination showed that there was not a proliferation of protein components but, rather, a considerable change in the concentration of existing protein fractions. Several fractions, assumed to be components of the reserve or storage proteins, showed a quantitative depletion correlated in time with the loss of the protein bodies. Quantification of the soluble proteins during germination showed a 32% decrease per radicle tip over a 67-hour period, but when expressed on a per cell basis a 32% increase was noted. A dramatic increase in protein synthesis, as measured by ³H-leucine incorporation, was initiated after 36 hours' exposure to germinative conditions, that is, at the beginning of radicle protrusion. A time-ordered sequence of events was noted for the depletion of the protein bodies and their assumed reserve components, suggesting their ultimate utilization in new protein synthesis.     

The incorporation of [14C] lysine into the protein of the guinea pig central auditory system

The incorporation of [¹⁴C]lysine into various brain proteins was studied. The proteins of different areas of the auditory system and cortical subcellular fractions were analysed using a disc electrophoretic technique that allows both protein and radioactivity assays along the gels. The highest level of incorporation was found in the mid brain nuclei, particularly the inferior colliculus, and was lowest in the auditory cortex proteins. This was true for both saline soluble proteins and proteins solubilized by Triton X-100 treatment. Of the subcellular fractions, the highest level of activity was found in the microsomal fraction. Considerable radioactivity was also found in the proteins isolated from the synaptosome-rich fraction. Of particular interest in this fraction was a slow migrating protein band which was soluble in Triton X-100, had a high specific activity, and appeared to be synaptosome specific. These observations are in concurrence with the hypothesis that the nerve ending contains protein synthesizing machinery.     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

Immunodiffusion analysis of water-soluble rat bone marrow antigens

Antisera were obtained against six electrophoretic fractions of the rat bone marrow extract. With their help, 18 tissular antigens and 11 antigens immunologically similar to the blood serum proteins were revealed in the rat bone marrow. All tissular antigens are divided in five groups by the degree of organ specificity: 1) bone marrow organospecific antigens (4 antigens), 2) antigens present in the bone marrow, spleen and lung extracts (2), 3) "granulocytic" antigens (4), 4) antigens common for many rat organs, but not found in the extracts of blood formed elements, skeletal muscle, heart, brain, eyes (3), 5) antigens present in all the organs studied (5). The bone marrow organospecific antigens may be specific antigens of hemopoietic cell precursors. The possibility of utilization of antisera against the bone marrow water soluble proteins for labelling hemopoietic cells of different lines of differentiation is discussed.     

Study of soluble lipoprotein in rat liver mitochondria

1. A water-soluble lipoprotein was isolated and purified from osmotically shocked preparations of rat liver mitochondria by using a technique of Sephadex-sandwich disc electrophoresis. 2. The purified lipoprotein migrates as a distinct sharp zone in high-resolution electrophoretic systems, indicating high degree of purity. 3. The lipoprotein resembles mitochondrial membranes with respect to lipid composition and lipid/protein ratio. 4. The lipoprotein and its apoprotein fraction obtained by delipidization at -18 degrees C to -20 degrees C have common properties with respect to their fluorescence spectra, instability to storage and electrophoretic mobility. 5. The purified lipoprotein has an excitation maximum at 325nm and a fluorescence maximum at 418nm. 6. Storage at 4 degrees C for 4 days or repeated freezing and thawing results in 15-30% decrease in electrophoretic mobility. 7. The patterns of incorporation in vitro of [1-(14)C]leucine into proteins of the soluble lipoprotein and of mitochondrial membrane of isolated rat liver mitochondria suggest a probable precursor role for the apoprotein in the formation of mitochondrial membrane protein. 8. Lipoprotein preparations isolated from mitochondrial fractions of rat kidney, brain and heart and of chicken and mouse liver resemble closely that obtained from rat liver mitochondria, suggesting that the soluble lipoprotein could be a distinct entity of mitochondrial origin.     

Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.     

The disc electrophoretic separation of proteins from various parts of the guinea pig brain

—Disc electrophoresis was used to study the saline-soluble and detergent-soluble proteins of various parts of the auditory pathway in the guinea pig brain. The five areas studied were the cochlear nucleus, olivary complex, inferior colliculus, medial geniculate and the auditory cortex. A study was also made of the proteins extracted from subcellular fractions obtained from guinea pig cerebral cortex. The electrophoretic patterns showed small but significant differences in the five areas of the brain, both in the fast-running acidic proteins and in the very slow-moving proteins. The higher levels of the auditory pathway, to which the more complex information processing and storage is normally attributed, showed more complex electrophoretic patterns than the lower areas. Gross differences also occurred in the patterns of both the saline-soluble and T-X-100-soluble proteins of the subcellular fractions obtained by gradient density centrifugation. The simplest patterns were obtained from the myelin-rich fraction and the mitochondrial fraction whilst the most complex patterns were given by the proteins of the nerve ending fraction and the cell soluble fraction.     

Composition of rat serum proteins under the effect of acetaldehyde and ethanol

Concentration and composition of rat serum proteins have been studied both under the isolated acetaldehyde effect and against a background of the action of acute and chronic intoxication by ethanol. Acetaldehyde evoked an increase in the concentration of the total serum proteins, the value of this parameter remaining unchanged under the ethanol effect. A comparison of ethanol and acetaldehyde effects gives ground to suppose that the quantitative redistribution of blood serum proteins which observed under ethanol intoxication is to a considerable extent a result of the acetaldehyde effect and is determined by aggregation of proteins.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.