Soluble Fraction Of Rat Brain Research Articles

Specific binding of tubulin to a guanine nucleotide-binding inhibitory regulatory protein in adenylate cyclase system, N i

A protein was isolated from the soluble fraction of rat brain by affinity chromatography with Sepharose to which guanine nucleotide-binding inhibitory regulatory protein in adenylate cyclase system, N i, was immobilized. The molecular weight of this protein, specifically bound to the N i-affinity column, was estimated as 54,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Alternately prepared tubulin also bound to the N i-affinity column. The amino acid compositions of these proteins were also identical. It is strongly suggested that this N i-binding cytosolic protein is tubulin.

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease.

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.

31] Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from rat brain

Ca 2+- activated, phospholipid-dependent protein kinase (protein kinase C) is a new species of protein kinase that is activated by diacylglycerol in the presence of both membrane phospholipid and physiological concentrations of Ca 2+ . Protein kinase C was originally found as an inactive enzyme that could be activated by limited proteolysis with Ca 2+ dependent neutral thiol protease. The enzyme was subsequently shown to be alternatively activated in a reversible manner by attachment to membrane phospholipid in the presence of Ca 2+ . Further analysis revealed that a small amount of diacylglycerol dramatically increases the affinity of the enzyme for Ca 2+ and for phospholipid, and thereby causes activation of the enzyme at physiological concentrations of Ca 2+ . This chapter describes the purification and properties of protein kinase C from a rat brain soluble fraction. The enzyme lacks tissue and species specificities, in the physical and kinetic properties that are described in this chapter.

Clorgyline and deprenyl insensitive monoamine oxidase in rat brain soluble fraction

A substantial percentage of monoamine oxidase activity was found in the soluble fraction of rat brain homogenates. The enzyme was not inhibited in vivo by high doses of clorgyline and deprenyl (12 mg/kg for 2 hr). Monoamine oxidase activity associated with the soluble fraction did not show the usual biphasic dose-response kinetics with clorgyline when kynuramine was used as the substrate. The present data show that there may be a new form of monoamine oxidase associated with the soluble fraction of the cell which has properties different from the two well-known monoamine oxidases A and B.

Total tubulin and its aminoacylated and non-aminoacylated forms during the development of rat brain.

The amount of total tubulin in the soluble fraction of rat brain was measured by a method based on the purification of tubulin previously labeled by incorporation of [14C]tyrosine in the C terminus of its alpha-chain. The tubulin content decreased from 2.01 to 1.30 nmol/mg protein when the animals passed from 4 to 30 days of age and then remained practically constant. The amounts of aminoacylated and non-aminoacylated tubulin present in the soluble brain extracts were determined from the incorporation of [14C]tyrosine into the free acceptor sites of tubulin preparations, that were preincubated without carboxypeptidase A or with this enzyme to eliminate tyrosine and phenylalanine from the C terminus of the alpha-chain of tubulin. The values obtained were corrected for the inactivation of tubulin to accept [14C]tyrosine that occurred during the isolation and incubation of the soluble fractions. The ratio non-aminoacylated/aminoacylated tubulin increased from 1.62 +/- 0.03 in the 4-day-old rats to 2.11 +/- 0.17 in the 120-day-old rats. The aminoacylatable tubulin, that is the sum of aminoacylated plus non-aminoacylated tubulin, decreased from 1.71 to 0.75 nmol/mg protein from 4-day-old to 30-day-old rats respectively and then remained practically constant. The amount of aminoacylatable tubulin is lower than that of total soluble tubulin. Therefore there is a fraction of tubulin that is unable to accept tyrosine. This non-aminoacylatable tubulin fraction increases with the age of the animal so that in the 120-day-old rats this tubulin species accounts for 48% of the total soluble tubulin.

A NOVEL ENZYME IN RAT BRAIN CONVERTING β‐ENDORPHIN INTO METHIONINE ENKEPHALIN: AFFINITY CHROMATOGRAPHY AND SPECIFICITY

Abstract— Methionine enkephalin (met‐enk), an endogenous opioid, commonly occurs in sequences of lipotropins (LPH) and endorphins, implying a possible biosynthetic pathway for this pentapeptide. In search of the enzyme which generates met‐enk from human β‐endorphin [LPH(61‐91), β‐end], it was found that the soluble fraction of rat brain contained such activity. The enzyme was purified by ammonium sulfate fractionation (50‐80% saturation), ion‐exchange chromatography on DEAE‐Sephadex A‐50, and affinity chromatography for which LPH(64‐67) functioned as affinity ligand and eluant. The final preparation was essentially free of other peptidases, such as kininase, met‐enk degrading and post‐proline cleaving activities. Studies on specificity revealed that the enzyme selectively cleaved the Met‐Thr bond in both LPH(64‐67) and β‐end. It is possible that this enzyme participates in the biosynthesis of met‐enk in vivo.

FRACTIONATION OF SOLUBLE BRAIN GLYCOPROTEINS BY AFFINITY AND HYDROXYLAPATITE CHROMATOGRAPHY

Abstract—A separation of soluble brain proteins and Con A‐binding glycoproteins by chromatography on calcium hydroxylapatite in the presence of SDS is described. Seventeen Coomassie Blue‐stained bands were detected by polyacrylamide gel electrophoresis in SDS of Con A‐binding glycoproteins of the soluble fraction of rat brain: 16 of these were found by in vivo uptake of [3H]fucose to be fucosylglycoproteins. Hydroxylapatite chromatography yielded several glycoprotein pools, each of which was shown by gel electrophoresis to contain between 4 and 8 individual glycoproteins. Such pools were enriched in [36H]fucose relative to the brain soluble fraction by factors of between 6 and 21. Preliminary experiments demonstrate that this method is also applicable to the fractionation of membrane‐bound glycoproteins.

Cyclic AMP-stimulated protein kinases at brain synaptic junctions.

We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems.

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37°C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480000. Lubrol PX, hydroxylamine, or NaN 3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN 3 plus catalase, N- methyl-N′- nitro-N- nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn 2+ for its activity; the maximum activity was observed at 3–5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(β,γ-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN 3 plus catalase or N- methyl-N′- nitro-N- nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.

Two integral antigens shared by the synaptic membranes and endoplasmic reticulum of rat brain: Separation by differential extraction

Two major brain membrane antigens designated X1 and X2 were found to be shared by rat brain synaptic membranes (RSM) and rat brain endothelial reticulum (RBER) by immunodiffusion analyses with IgG solutions prepared from the sera of rabbits hyperimmunized with RSM and RBER. The antisera were absorbed with the soluble fraction of rat brain (RBE) which removed antibodies directed against antigens in the soluble fraction of brain. Antigens X1 and X2 are considered to be integral components of the RSM and RBER membrane matrixes because they were not removed from the membranes by sequential washing with sodium salts but were extracted with Triton X-100 or butanol. Sequential treatments of RSM and RBER with different concentrations and volumes of Triton X-100 yielded Tx1a, Tx1b and Tx2 extracts; Tx1 solutions contained antigens X1 and X2 while Tx2 solutions contained only antigen X2. Treatment of RSM or RBER with butanol rendered both antigens soluble in water. The concentrated aqueous solutions of X1 and X2 were stable for several months in 0.5% Triton X-100. Anti-X1 antisera were prepared by absorbing anti-RSM sera with RBE and RSM-Tx2 extracts.

Release of tyrosine from tyrosinated tubulin. Some common factors that affect this process and the assembly of tubulin

An enzyme system present in the soluble fraction of rat brain homogenate catalyzes the incorporation of tyrosine into the carboxyl end of the a-subunit of tubulin [ 1,2] . The system does not require nucleic acids [3]. Results from studies on the incorporation of [r4C] tyrosine into brain tubulin in animals whose protein synthesis was inhibited by cycloheximide indicated that a similar system operates in vivo [4]. The present work deals with an activity found in the soluble fraction of rat brain that determines the release of the C-terminal tyrosine from tyrosinated tubulin. Every previously known inhibitor or activator of assembly of tubulin we tested also affected the release of tyrosine when assayed in similar conditions. The relationship of the activity reported here with a similar one previously described [S] for which was claimed that ADP and Pi were required is discussed.

Catecholamine Binding Macromolecule in Soluble Fraction of Rat Brain

A macromolecule having a high binding affinity to norepinephrine was isolated from the soluble fraction of rat brain, the partially purified macromolecule was prepared by extraction of the brain with isotonic sucrose containing 3.3 mM CaCl2, ultracentrifugation, 35–50% ammonium sulfate precipitation and DEAE-cellulose chromatography. DEAE-cellulose chromatography revealed that the macromolecule in the soluble fraction was separated in at least 4 fractions, 2 of which had both norepinephrine and cyclic AMP binding activity. A Scatchard plot indicated that a partially purified macromolecule showed 2 apparent binding affinity constants of 4.13×10−7 M and 3.70×10−6 M to norepinephrine. The effect of chemical or physical treatments on specific binding of the macromolecule to norepinephrine was studied in comparison with cyclic AMP binding to the macromolecule. Trypsin treatment caused partial loss of norepinephrine binding activity, while cyclic AMP binding activity was completely lost. Heat treatment resulted in a complete disappearance of cyclic AMP binding activity, while norepinephrine binding activity was only slightly decreased. Dibenamine treatment caused a marked decrease both of norepinephrine binding activity and that of cyclic AMP. Dichloroisoproterenol had no effect on the binding activities. Cyclic AMP inhibited clearly norepinephrine binding to the macromolecule, while norepinephrine inhibited cyclic AMP binding only slightly. Norepinephrine binding to the macromolecule was essentially dependent on its catechol structure. The entity of the macromolecule is discussed on the basis of the findings.

Isolation and properties of a soluble chondroitin sulfate proteoglycan from brain

A proteoglycan in which the glycosaminoglycans are predominantly chondroitin sulfate has been isolated from the soluble fraction of rat brain by ion exchange chromatography and gel filtration. Glycoprotein oligosaccharides are also present, and result in adsorption of the proteoglycan by Concanavalin A-Sepharose. The proteoglycan-glycoprotein complex eluted from the affinity column by α-methylglucoside floats near the top of a cesium chloride density gradient run under dissociative conditions (4 M guanidine), but after β-elimination of the chondroitin sulfate polysaccharide chains from their low buoyant density glycoprotein complex they sediment to the bottom of the gradient. These results suggest that relatively few polysaccharide chains are covalently linked to a large protein core in the dissociated chondroitin sulfate proteoglycan “subunit” from brain, and that the proteoglycans are closely associated with soluble glycoproteins.

Regulation of transsynaptically elicited increase of 3':5'-cyclic AMP by endogenous phosphodiesterase activator.

Experiments with cold exposure confirmed previous studies indicating that the endogenous protein acitvator of phosphodiesterase (PDEA) isolated by Cheung participates in the in vivo regulation of 3':5'-cyclic adenosine monophosphate (cAMP) in adrenal medulla. This activator of cAMP phosphodiesterase (PDE) (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) is present in the particulate as well as the soluble fractions of rat brain. It was found that a purified cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), in the presence of ATP and cAMP, stimulates 3-fold the release of PDEA from the particulate fraction of rat brain and adrenal medulla. The substrate for this phosphorylation could be either a membrane protein that binds PDEA or PDEA itself. In vivo evidence, however, obtained by injecting rats intraventricularly with [gamma-32P]ATP, indicates that the PDEA does not contain radioactive phosphate in its structure. Also, PDEA could not be phosphorylated by protein kinase in vitro. The following mechanism is postulated: when the intracellular content of cAMP increases it activates a protein kinase which phosphorylates a PDEA-binding membrane protein and releases PDEA. In turn this binds to activator-deficient high Km PDE and decreases its Km to facilitate the hydrolysis of the increased concentration of cAMP.

Cysteine sulfinic acid decarboxylase in rat brain: Effect of vitamin B 6 deficiency on soluble and particulate components

Cysteine sulfinic acid decarboxylase activity is not affected when measured in vitro in the presence of pyridoxal phosphate in the brains of vitamin B 6-deficient rats. Activity of this enzyme was not detectable in the brains of vitamin B 6-deficient animals when assayed in the absence of pyridoxal phosphate. The activity of cysteine sulfinic acid decarboxylase in crude particulate and soluble fractions of rat brain reflects the distribution of the enzyme in vivo and the distribution of endogenous B 6 vitamers when assayed in the presence and absence of vitamin B 6. This study indicates that virtually no taurine is synthesized by any component of brain when the animal is subjected to a deficiency of vitamin B 6.

Methylenetetrahydrofolate reductase in the rat central nervous system: Intracellular and regional distribution

Methylenetetrahydrofolate reductase and methyltetrahydrofolate-homocysteine methyltransferase were found to be localized in the soluble fraction of rat brain. They are clearly separated from serine hydroxymethyltransferase and the glycine cleavage complex which are localized in the mitochondria in this tissue. Hence, although the primary, if not the only, site of 5,10-methylenetetrahydrofolate formation in brain appears to be the mitochondrion, the utilization of this compound for 5-methyltetrahydrofolate synthesis and utilization of the latter compound for methylation of homocysteine occurs in the cytosol. Parallel experiments with rat liver confirmed that the reductase and homocysteine methyltransferase of this tissue are also localized in the soluble fraction, while the hydroxymethyltransferase is about evenly divided between mitochondria and cytosol. However, in liver (but not in brain) the reductase activity of the supernatant fraction is only partially expressed unless the fraction is dialyzed. We have found that this phenomenon, which initially suggested the occurrence of an endogenous inhibitor in liver extracts [Ordoñez and Wurtman (1973) J. Neurochem., 21, 1447–1455] , is due to loss of the product (5, 10-methyl-enetetrahydrofolate) of the reductase assay via its oxidation by methylenetetrahydrofolate dehydrogenase, in combination with the NADP present in undialyzed extracts. All regions of the rat central nervous system tested contained methylenetetrahydrofolate reductase activity. Regional variations were observed however, with an almost threefold difference between the specific activities of the highest and lowest ranking regions Comparison of the rank order of 12 regions tested with respect to reductase specific activity (this study) and methyltetrahydrofolate-tryptamine N-methyltransferase specific activity [Mandell, Knapp, and Hsu (1974) Life Sci., 14, 1–17] shows a high positive correlation ( r = 0.916) between these activities in the selected regions.

MODIFICATIONS IN SOLUBLE PROTEIN KINASE AND CYCLIC‐AMP BINDING CAPACITY OF DEVELOPING RAT BRAIN

Abstract—Histone and casein phosphoprotein‐kinase activities were determined in rat brain soluble fraction at various stages of development.Cyclic AMP‐independent or basal histone kinase activity increased, whereas cyclic AMP‐dependent activity decreased in whole soluble fraction with the age. On the contrary, whole soluble cyclic AMP‐dependent and ‐independent casein kinases activities did not show any difference during development.The percentage of activation by cyclic AMP of histone kinase activity and [3H] cyclic‐AMP binding activity in the soluble fraction decreased markedly during development.By DEAE‐cellulose chromatography the histone kinase was separated mainly into 4 peaks; the fourth peak was strongly stimulated by cyclic AMP. Stimulation by cyclic AMP was higher in the 4‐day‐old rat brains than in the 9‐ and 30‐day‐old. In the 9‐day‐old rats the ratio of cyclic AMP‐dependent histone kinase in respect to the cyclic AMP‐independent enzyme was higher than in 4‐ and 30‐day‐old rats.Casein kinase activities in the brains of 9‐ and 30‐day‐old rats were separated by DEAE‐cellulose chromatography into three peaks of which the third one was stimulated by cyclic AMP. Little, if any, difference was observed for casein kinase during the development. These results suggest that brain histone and casein kinase are different enzymes:

Ascorbic acid-like effect of the soluble fraction of rat brain on adenosine triphosphatases and its relation to catecholamines and chelating agents

The inhibitory effect of the soluble fraction of rat brain on the Na +, K +-ATPase, Mg 2+-ATPase and K +- p-nitrophenylphosphatase activities of rat brain subcellular particles and its antagonization by catecholamines have been studied. The soluble fraction could be replaced by l-ascorbic acid in the process, but dehydroascorbic acid or cytoplasmic reducing agents such as l-cysteine and l-glutathione did not show the same effect. Catecholamines could be replaced by chelating agents; EDTA, EGTA, o-phenanthroline and α,α'-dipyridyl prevented the inhibition by the soluble fraction and by l-ascorbic acid. The inhibition of the enzyme activities was not prevented by catecholamines or chelators when they were added after preincubation of the enzyme preparation in the presence of soluble fraction or of l-ascorbic acid. The effect of EGTA was eliminated by an excess of Ca 2+ and the effect of EDTA by an excess of Ni 2+. Excesses of Ca 2+, Mn 2+ and Co 2+ did not influence the effect of EDTA. It is supposed that a reoxidizable heavy metal bound to the membrane structures is responsible for the inhibitory effect of the soluble fraction and of l-ascorbic acid. Catecholamines may act by chelating this metal. The possible role of iron in the phenomenon has been investigated and discussed.

Studies on Guanylate Cyclase: A NEW ASSAY METHOD FOR GUANYLATE CYCLASE AND PROPERTIES OF THE CYCLASE FROM RAT BRAIN

Abstract A new rapid and reliable assay for guanylate cyclase was established, which employs a simple procedure to isolate guanosine 3' : 5'-monophosphate with neutral aluminum oxide and Dowex 1 formate columns. Using this assay guanylate cyclase was purified about 30-fold from rat brain soluble fraction by ammonium sulfate fractionation, followed by calcium phosphate gel adsorption and elution. Mn2+ was essential for the activity and neither Ca2+ nor Mg2+ could substitute Mn2+. In the presence of a low concentration of Mn2+ the enzyme was stimulated severalfold by Ca2+. The topographical distribution of both guanylate cyclase and guanosine 3' : 5'-monophosphate phosphodiesterase in rat brain was also described.

Colchicine-binding protein in rat brain: Biochemical consistency and immunological discrepancy

The colchicine-binding protein in the soluble fraction of rat brain was characterized biochemically and immunologically. On the basis of molecular size and charge, the tight binding of colchicine appeared to be the property of a single macromolecule. Colchicine-binding protein was purified from the soluble fraction of rat brain by the ammonium sulfate fractionation-DEAE chromatography method and also by precipitation with vinblastine sulfate. Both preparations interacted similarly with antibody but only the latter elicited a humoral immune response in rabbits. The possible significance and causes of the immunological discrepancy are discussed.