Identification, purification and physico-chemical properties of neurospecific alpha 1-globulin]

The partially purified and concentrated 500-600-folds protein fraction has been obtained from human brain extract. This protein fraction was used for the immunization of rabbits. The corresponding anti-sera have the potency to detect the brain specific alpha 1-globulin, which is not identical to known cytoplasmatic brain specific protein. These antisera were used for the control of the antigen purification procedure which included ion-exchange, affinity and hydropho'ic chromatography, gel-filtration and isochromatofocusing. The antigen, purified to the homogeneity, has the electrophoretic mobility of the alpha 1-globulins, M(r) = 110-10 kD, and isoelectric point at pH 2.9-3.1.     

Beta 2 subunits of sodium channels from vertebrate brain. Studies with subunit-specific antibodies

The sodium channel purified from rat brain is composed of three subunits: alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000). alpha and beta 2 subunits are linked through disulfide bonds. Procedures are described for preparative isolation of the beta 1 and beta 2 subunits under native conditions. Pure beta 2 subunits obtained by this procedure were used to prepare a specific anti-beta 2 subunit antiserum. Antibodies purified from this serum by antigen affinity chromatography recognize only disulfide-linked alpha beta 2 complexes and beta 2 subunits in immunoblots, and immunoprecipitate 32P-labeled alpha subunits of purified sodium channels having intact disulfide bonds, but not those of sodium channels from which beta 2 subunits have been detached by reduction of disulfide bonds. These antibodies also immunoprecipitate 89% of the high affinity saxitoxin-binding sites from rat brain membranes, indicating that nearly all sodium channels in rat brain have disulfide-linked alpha beta 2 subunits. Approximately 22% of beta 2 subunits in adult rat brain are not disulfide-linked to alpha subunits. Anti-beta 2 subunit antibodies are specific for sodium channels in the central nervous system and will not cross-react with sodium channels in skeletal muscle or sciatic nerve. The brains of a broad range of vertebrate species, including electric eel, are shown to express sodium channels with disulfide-linked alpha beta 2 subunits.     

Developmental changes in translatable mRNAs for the cerebral enolase isozymes alphaalpha and gammagamma

Using the rabbit reticulocyte cell-free translation system we have estimated during ontogenesis the proportions of in vitro translatable alpha and gamma brain enolase mRNAs, which are two minor mRNA species. No polypeptide precursor to these enzyme subunits appears to be synthesized during translation in vitro. During brain development, the changes in translatable alpha and gamma mRNA content seem to parallel those of the corresponding antigens. The proportion of each of the enolase mRNAs is highest in adult mouse brain. Mechanisms controlling alpha and gamma antigen expression are discussed. In order to prepare the specific cDNA probes, purification of alpha and gamma mRNAs was undertaken.     

Immunoenzymatic detection of specific brain antigens as a criterion of the permeation of blood-brain barrier in rats with acute cerebral ischemia

EIA detection system for the measurement of alpha 2 M globulin and GFAP antigen has been developed. The limit of the sensibility was only 1 ng/ml for alpha 2M and 0.8 ng/ml for GFAP. The system was used for the studies of the penetration through the blood-brain barrier in rats with experimental acute brain ischemia. The measurement of alpha 2M and GFAP antigens by EIA technique 16-20 hours after the occlusion of the carotid artery has revealed disturbances in the blood-brain barrier permeability for specific brain proteins. The method is recommended for indirect evaluation of the blood-brain barrier functional disorders.     

Characterization of a novel Le(x)-active ganglioside from chick intestinal tissues recognized by murine monoclonal antibody 188C1.

A monoclonal antibody, 188C1, raised against skin tissue from the back of bullfrogs (Rana catesbeiana) was found to recognize a common antigen in neural and intestinal tissues of chicken (Fujita, S. (1989) in Biological Transduction Mechanisms (Kasai, M., Yoshioka, T., and Suzuki, H., eds) pp. 159-177, Japan Scientific Societies Press, Tokyo Japan). The 188C1 antigen was isolated from chick intestinal tissues and characterized as a novel ganglioside by means of Q-Sepharose and Iatrobeads column chromatography, and chemical, immunochemical, and immunohistochemical analyses. The chemical structure was as follows: Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1'Cer Fuc alpha 1-3 NeuAc alpha 2-3 This represents a novel hybrid structure of type 2 Le(x) epitope and GM1 ganglioside core structure, designated as Le(x)-GM1. Monoclonal antibody 188C1 reacted strongly with Le(x)-GM1 on thin layer chromatography, but its reactivity was greatly reduced when sialic acid was removed from the antigen. This indicated that the internal sialic acid residue might participate in antigenicity of the Le(x) determinant. In addition to 188C1, a more specific antibody reacting with Le(x)-GM1 but not with asialo-Le(x)-GM1 was raised by immunizing a rabbit with Le(x)-GM1. TLC/enzyme immunostaining using this specific antibody showed the presence of Le(x)-GM1 in chick intestinal tissue, but not in chick brain.     

Immunoenzyme determination of neurospecific antigen 10-40-4 in human and animal tissue and organ extracts

IEA of brain specific antigen 10-40-4 in human and animal organs and tissues was elaborated. The method permits measuring the concentration of the antigen within the range from 1 to 120 ng/ml. The use of the method made it possible to confirm brain specificity of protein 10-40-4, since its brain content is 1500 to 2000 times higher than in other organs, and to estimate the percentage of cross-reaction between antigenic determinants of brain specific antigen 10-40-4 from different species of animals.     

Structural analysis of a glycosylphosphatidylinositol glycolipid ofLeishmania donovani

A glycosylphosphatidylinositol (GPI) glycolipid antigen recognized by sera from patients with visceral leishmaniasis was isolated from Leishmania donovani promastigotes. The carbohydrate moiety was cleaved from the lipid part by digestion with specific phosphatidylinositol phospholipase C. After separation, structural analysis was carried out on the phosphorylated inositol oligosaccharide and the alkylacyl glycerol. The following major structures were found: [formula: see text] The presence of the conserved sequence Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN-PI of glycosyl phosphatidylinositol protein anchors in this antigen may be consistent with a precursor role of Leishmania glycosyl phosphatidylinositol anchored proteins for this glycolipid.     

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

Location of sialoglycoconjugates containing the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups in the rat hippocampus and the effect of aging on their expression.

The histochemical distribution of sialoglycoconjugates in the CA1 region in the hippocampus formation of 9-week-old rats and 30-month-old rats was examined using electron microscopy in combination with two lectins, Maackia amurensis lectin, specific for Sia(alpha)2-3Gal, and Sambucus sieboldiana agglutinin, specific for Sia(alpha)2-6Gal. Each lectin stained the plasma membranes of pyramidal cells, indicating that the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups were expressed on their plasma membranes. These lectins also bound to synapses in the stratum lacunosum molecular. The staining intensity of the lectins in the synapses in these layers was downregulated in the 30-month-old rats. These results indicated that both the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups are expressed on these synapses and that the expression of these sialyl linkages decreases in the aged brain     

Mapping the orientation of an antigenic peptide bound in the antigen binding groove of H-2Kb using a monoclonal antibody.

The major histocompatibility complex class I molecules are receptors for intracellular peptides, both of self and non-self origin. When non-self peptides (eg., pathogen derived) are bound to the class I molecules, they form ligands for T cell receptors resulting in antigen specific lysis of the infected cells by cytotoxic T lymphocytes. Therefore, an understanding of the process of antigen recognition requires the precise definition of the structural features of the bimolecular complex formed by a single well defined antigenic peptide bound to the class I molecule. A strategy using antibodies was developed to probe the structural features of the H-2Kb containing a defined peptide in the antigen cleft. We report that the binding surface area of a Kb specific monoclonal antibody (28-13-3s) includes residues in the alpha 1 (Gly56 and Glu58) and alpha 2 (Trp167) helices of Kb thus, binding across the antigen binding groove. When cells treated with the antigenic peptide of vesicular stomatitis virus, N52-59, and its alanine substituted analogs were tested for 28-13-3s binding, it was found that position 1 of the peptide also forms a part of the antibody binding site. This finding strongly supports the positioning of the N-terminus of N52-59 proximal to pocket A, thus, assuming an orientation parallel to the alpha 1 helix.     

Isolation of an endogenous clonidine-displacing substance from rat brain

An endogenous substance which specifically displaces clonidine, yohimbine and rauwolscine from rat brain alpha 2-adrenergic receptors, has been isolated. The new compound, designed clonidine-displacing-substance (CDS), has been partially purified by ion exchange chromatography, zone electrophoresis and high performance liquid chromatography (HPLC). CDS binds specifically to alpha 2-adrenergic receptors by competing with either alpha 2-adrenergic agonists or alpha 2-antagonists, but has no effect on the specific binding of [3H]prazosin to alpha 1-adrenergic receptors in rat brain membranes. In the course of isolation, CDS was shown to be neither the endogenous neurotransmitter (-)norepinephrine (NE) nor the guanyl nucleotide GTP which lowers the specific binding of alpha 2-agonists to the alpha 2-adrenergic receptors.     

Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule

A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as alpha 2-8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).     

Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer) and to its 9-O-acetylated derivative

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides. In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study. One is the ganglioside NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GT3) and the other is a 9-O-acetylated derivative of GT3). This derivative was purified from 10-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-O-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5. These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18B8 in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalcohol chain. Antibody 18B8 does not bind to 9-O-acetylated GT3 or GT3 oxidized by sodium periodate.     

THE NATURE OF THE TWO PROTEINS OF BRAIN SPECIFIC ANTIGEN 14-3-2

The three isoenzymes of rat brain enolase (2-phospho d -glycerate hydrolase EC 4.2.1.11.) χχ, χγ and γγ were separated by ion-exchange chromatography and were tested for reaction with an antiserum against brain specific antigen 14-3-2. This monospecific antiserum affects the enolase activity of only the χγ and γγ isoenzymes. Immunoelectrophoretic experiments show that the two proteins which react as 14-3-2 both contain γ enolase subunits, and one of these also contains χ enolase subunits. It is concluded that the 14-3-2 antigen and the γ enolase subunit are identical, and that the two proteins which react immunologically as 14-3-2 are the χγ and γγ enolase isoenzymes.     

Brain adrenoreceptors in rats with inherited arterial hypertension due to emotional stress

For the study of genetic and physiological mechanisms of inherited stress-sensitive arterial hypertension, specific binding of ligands of alpha 1-, alpha 2- and beta-adrenoceptors was measured in 2 strains of rats: Wistar normotensive and ISSAH rats (rats with inherited stress-sensitive arterial hypertension). The maximal binding sites (Bmax) and apparent dissociation constants (Kd) were studied with the alpha 1-adrenergic antagonist 3H-prazosin, alpha 2-adrenergic agonist 3H-clonidine and 3H-dihydroalprenolol, a beta 1-receptor antagonist. Four brain regions were investigated: frontal cortex, hypothalamus, pons and medulla oblongata. In comparison with normotensive controls, hypertensive rats had significantly greater density of the alpha 1-adrenoceptors in the medulla oblongata. However, the number of hypothalamic alpha 1-adrenoceptors was significantly reduced in these animals. The same significantly lower alpha 2-adrenoreceptor density was found in the hypothalamus and the pons, and lower, beta-adrenoceptors density in the medulla oblongata. It was concluded that brain adrenoceptors are involved in the mechanisms of development of inherited stress-sensitive hypertensive syndrome.     

Early cortical plate specific glycoprotein in a marsupial species belongs to the same family as fetuin and alpha 2HS glycoprotein

Two related glycoproteins, fetuin in species of the order Artiodactyla (cattle, sheep, pig) and alpha 2HS glycoprotein in the human [(1987) Cell Tissue Res. 248, 33-41] have a very specific distribution in the developing brain. We have isolated and determined the first 15 N-terminal residues of a similarly distributed glycoprotein in the developing brain of the tammar wallaby (Macropus eugenii). The degree of homology is the same between wallaby glycoprotein and alpha 2HS glycoprotein as between fetuin and alpha 2HS glycoprotein (46%). Antibodies made to synthetic peptides of fetuin were used to identify the wallaby glycoprotein. A polyclonal antibody to the purified glycoprotein was used for immunocytochemical identification of brain cells positive for this protein.     

Presynaptic localization of histamine H3-receptors in rat brain.

The localization of histamine H3-receptors in subcellular fractions from the rat brain was examined in a [3H] (R) alpha-methylhistamine binding assay and compared with those of histamine H1- and adrenaline alpha 1- and alpha 2-receptors. Major [3H](R) alpha-methylhistamine binding sites with increased specific activities ([3H]ligand binding vs. protein amount) were recovered from the P2 fraction by differential centrifugation. Minor [3H](R)alpha-methylhistamine binding sites with increased specific activities were also detected in the P3 fraction. Further subfractionation of the P2 fraction by discontinuous sucrose density gradient centrifugation showed major recoveries of [3H](R)alpha-methylhistamine binding in myelin (MYE) and synaptic plasma membrane (SPM) fractions. A further increase in specific activity was observed in the MYE fraction, but the SPM fraction showed no significant increase in specific activity. Adrenaline alpha 2-receptors, the pre-synaptic autoreceptors, in a [3H] yohimbine binding assay showed distribution patterns similar to histamine H3-receptors. On the other hand, post-synaptic histamine H1- and adrenaline alpha 1-receptors were closely localized and distributed mainly in the SPM fraction with increased specific activity. Only a negligible amount was recovered in the MYE fraction, unlike the histamine H3- and adrenaline alpha 2-receptors.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.