IDENTIFICATION AND CHARACTERIZATION OF WATER SOLUBLE RAT BRAIN ANTIGENS I. BRAIN AND SPECIES SPECIFICITY

—Some new immunoelectrophoretic methods were applied to the study of brain and species specificity of water-soluble rat brain antigens. Five antigens were brain specific and one, a brain specific antigen, was species restricted.     

ANTIGEN—ANTIBODY CROSSED ELECTROPHORESIS OF WATER-SOLUBLE RAT BRAIN ANTIGENS

A method of separation by antigen-antibody crossed electrophoresis was applied to the study of water-soluble rat brain antigens. Five different rat brain preparations were used as antigens for immunization. The extract from Triton X-100 treated brains gave the best antibody response. An antiserum containing 27 precipitating antibodies was obtained. The preparation of antigens for immunoelectrophoresis was investigated. Treatment with demineralized water gave the highest number of antigen-antibody precipitates. Treatment with detergents and sonication gave a higher total protein yield, but the number of precipitates was unaffected.     

DETERMINATION OF BRAIN-SPECIFIC ANTIGENS IN SHORT TERM CULTIVATED RAT ASTROGLIAL CELLS AND IN RAT SYNAPTOSOMES

—The brain-specific antigens 14·3·2, GFA, A5, F3, D1, D2, D3 and C1 were quantitated in a short-term astroglial cell culture taken as a model of glial cells, and in synaptosomes, synaptosomal membranes and synaptic vesicles as neuronal material. Furthermore, the antigens were quantitated in newborn rat brain, as this served as the starting material for the cell culture. The membrane antigens C1, D1, D2 and D3 were absent from the cultured astroglia, indicating a neuronal origin for these antigens. C1 was enriched 3-fold in synaptosomes and synaptosomal membranes and more than 10-fold in synaptic vesicles indicating that this antigen might be a marker protein for nerve endings. The name Synaptin is introduced for this antigen. Conversely, the data on the antigens D1, D2 and D3 indicated that these antigens were not restricted to the synaptosomes although they were of neuronal origin. Trace amounts of the cathodal part of the heterogeneous cytoplasmic antigen 14·3·2 were present in the cell culture, possibly originating from a few contaminating neurons. The cytoplasmic antigens A5 and F3 were found both in the astroglial culture and in the synaptosomal fraction. F3, however, was found in low concentration in the synaptosomes and 3-fold enriched in newborn rat brain compared to rat brain from 35-day-old rats or to 21-day-old brain cell cultures. It was therefore regarded as a brain specific fetal antigen. The antigen GFA was highly enriched in the astroglial culture compared to whole brain and only trace amounts were found in the synaptosomal fraction supporting the astroglial origin of this antigen.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

PURIFICATION OF RAT BRAIN CHOLINE ACETYLTRANSFERASE1

Abstract— The purification of choline acetyltransferase (ChAc) has been hampered by the increasing instability of the enzyme in the course of purification. By working with a high concentration of protein and by adding glycerol to the enzyme, the stability was increased. The purification was performed by centrifuging twice, at low and high salt concentrations, precipitation by ammonium sulphate and chromatography on carboxymethyl–Sephadex, hydroxylapatite and Sephadex G 100. The final steps were performed by using chromatography on an immunoabsorbent; this consists of agarose-coupled gammaglobulins of antisera devoid of any activity against ChAc itself and directed against other proteins still present in the purest ChAc preparation achieved by conventional biochemical techniques. The purest rat brain ChAc preparation had a specific activity of 20 μmol/min/mg of protein after a 30,000-fold purification. The enzyme was not homogeneous in polyacrylamide gel electrophoresis performed either at pH 4.5 or with sodium dodecyl sulphate. Pure ChAc from rat brain would have a specific activity of approximately 100 μmol/min/mg of protein.     

SYNAPTIC AND NON-SYNAPTIC MITOCHONDRIA FROM RAT BRAIN: ISOLATION AND CHARACTERIZATION

Abstract— A method has been developed where by three distinct populations of metabolically active, well coupled and relatively pure mitochondria from rat brain may be prepared. Two mitochondrial populations are derived from synaptozomes and the third consists of 'free' (i.e. non-synaptic) mitochondria. These mitochondrial populations have been characterized with respect to both enzyme content and ability to oxidize substrates. The results indicate that these mitochondrial populations are heterogeneous with respect to maximal activities of certain enzymes concerned with the citric acid cycle and glutamate and 4-aminobutyrate metabolism as well as their ability to utilize various substrates. The data reported here also confirm that brain mitochondria are very heterogeneous and suggest that synaptic mitochondria may contain at least two sub-populations. The relations between the heterogeneity of brain mitochondria and the metabolic compartmentation of the citric acid cycle and related metabolites such as glutamate, aspartate and 4-aminobutyrate are briefly discussed in the light of two proposed models of metabolic compartmentation in the mammalian brain.     

ISOLATION AND STRUCTURAL STUDIES ON SYNAPTIC COMPLEXES FROM RAT BRAIN

A fraction enriched in synaptic complexes has been isolated from rat brain. The major structural elements of synaptic complexes after isolation are a sector of pre- and postsynaptic plasma membranes joined together by a synaptic cleft and a postsynaptic density (PSD) located on the inner surface of the postsynaptic membrane. On its outer surface, the postsynaptic membrane has a series of projections which extend about halfway into the cleft and which occur along the entire length of the PSD. Proteolytic enzymes at high concentrations remove the PSD and open the synaptic cleft; at low concentrations the PSD is selectively destroyed. By contrast, the structural integrity of the PSD is resistant to treatment with NaCl, EGTA, and low concentrations of urea. Pre- and postsynaptic membranes also remain joined by the synaptic cleft after NaCl, EGTA, or mild urea treatment. High concentrations of urea cause the partial dissociation of the PSD. We conclude that polypeptides are probably one of the major components of the PSD and that the structural integrity of the PSD depends on polypeptides because disruption of the covalent or hydrophobic bonding of these polypeptides leads to a progressive loss of PSD structure.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

THE EFFECTS OF LITHIUM ON ATPase ACTIVITY IN SUBCELLULAR FRACTIONS FROM RAT BRAIN

Abstract— The effects of lithium chloride in vitro and in vivo were investigated on Na-K ATPase and Mg ATPase activities in synaptic plasma membrane, mitochondrial and synaptic vesicle fractions prepared from rat brain. In vitro , lithium chloride (10⁻³-10⁻⁸ m ) had no effect on ATPase activity in any of the fractions studied. Lithium chloride given chronically by i.p. injection (30 mg/rat/day) for 9 days had little effect on synaptic plasma membrane ATPases. Dietary administration of lithium chloride (60 mmol/kg food) produced a small but significant increase in synaptic plasma membrane Mg ATPase activity after 3 weeks administration and mitochondrial Mg ATPase activity after 1 week. There was no effect on synaptic plasma membrane Na-K ATPase activity. Salt supplementation reduced the toxic effects of lithium administration and it is suggested that toxicity may account for some of the previously reported changes in synaptic membrane ATPases produced by lithium.     

PROPERTIES OF RAT BRAIN HISTIDINE DECARBOXYLASE

Abstract– The properties of histidine decarboxylase ( l -histidine carboxylyase EC 4.1,1.22) have been studied in a whole rat brain homogenate. Optimum pH depended upon substrate concentration; the variations of K _m and V _max were determined as a function of pH. pH values lower than 6.0 caused a loss of enzymic activity; activity was stable at pH values higher than 6.0. Enzyme activity was proportional to temperature in the range 30-45°C; temperature characteristic (μ) and Q₁₀ were determined and thermal inactivation was studied. Addition of pyridoxal 5'-phosphate increased enzyme activity. Dialysis of homogenates against phosphate buffer caused a partial loss of enzyme activity which could be restored by addition of the coenzyme to the incubation mixture. Enzyme activity was inhibited by α-methylhistidine and benzene and was unaffected by α-methyl DOPA. The properties correspond to those of a 'specific' histidine decarboxylase. However, the brain enzyme differs from the corresponding enzyme in peripheral tissues in the inability to achieve a total inhibition of activity by dialysis.     

ADENOSYLMETHIONINE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Adenosylmethionine decarboxylase from rat brain has been found to be similar to the same enzyme isolated from other rat tissues in regard to kinetic parameters, pH optimum, putrescine requirement, and subcellular location. Evidence is presented that pyridoxal phosphate is not the functional cofactor in enzymatic decarboxylation by the rat brain preparation. The capacity for spermidine synthesis in developing rat brain was determined by measurement of the activity of adenosylmethionine decarboxylase. The activity increased dramatically after 10 days of postnatal age. This increase occurred after the period of maximum nucleic acid synthesis, an observation which suggests that spermidine may have a role in the functional development of the brain.     

PROPERTIES OF RAT BRAIN NAD-KINASE

Abstract— NAD-kinase was purified from rat brain acetone powder according to the method of W ang and K aplan (1954). The acetate buffer supernatant showed only very low specific activity but was largely free of the factors that interfere with the enzyme assay. The Michaelis constants for both substrates were determined, the values were 0·5 m m for NAD and 4·0 m m for ATP. The optimal pH was 7·4 in tris-HCl buffer and the highest NAD-kinase activity was observed in the hyaloplasm fraction. NADH₂ inhibited the enzyme whereas NADPH₂ did not. Finally, the reversible inhibition of SH-binding compounds is described and the observed properties of rat brain NAD-kinase compared with the properties of NADP synthesizing enzymes from pigeon liver and rat liver.     

LOCALIZATION OF THE THY-1 ANTIGEN IN RAT BRAIN AND SPINAL CORD BY IMMUNOFLUORESCENCE

Abstract— The Thy-1 antigen of rat brain is a membrane glycoprotein of molecular weight 17,500. It was localized in sections of brain and spinal cord by indirect immunofluorescence using rabbit antisera raised against purified Thy-1 and fluorescein conjugated purified sheep F(ab')₂, anti-(rabbit IgG) antibody fragments. The specificity of the anti-(Thy-1) sera was tested by a quantitative indirect radioactive binding assay which is particularly useful for ascertaining the specificity of reagents used in immunohistochemical studies. Purified Thy-1 was used to absorb the anti-(Thy-1) sera for controls in the immunofluorescence experiments. Strong specific fluorescence was found throughout the gray matter of brain and spinal cord with lesser amounts in white matter. The nuclei of all neural cells and also myelin lacked fluorescence. Some of the large neurons contained weak cytoplasmic fluorescence, but the majority of the immunofluorescence was located in the neuropil of the brain and spinal cord. There was an indication that Thy-1 was associated with synaptic knobs due to its presence in synaptic glomeruli and its granular appearance around some neurons. An additional association with glial membranes could not be excluded.     

POLYADENYLIC ACID-CONTAINING RNA FROM RAT BRAIN SYNAPTOSOMES

Abstract— Synaptosomal RNA of rat brain was labelled in vivo by intracranial injection of tritiated uridine. The change in the specific activity of this material with time was similar to that of polysomal RNA. The percent of the radioactive synaptosomal RNA which bound to oligo(dT)-cellulose columns decreased with time after intracranial labelling. The percent of the total synaptosomal RNA which bound to oligo(dT)-cellulose was greater than that of polysomes. The length of the polyadenylate (poly(A)) sequence of synaptosomal RNA was approximately one-half that of polysomal RNA, and about the same as that from mitochondria. Investigation of synaptosomal RNA using sucrose gradients and polyacrylamide gel electrophoresis indicated that there were several distinct species present, and that they were similar to those from the mitochondria. The poly(A)-containing RNA isolated from synaptosomes stimulated the incorporation of radioactive leucine into TCA-precipitable material in a cell-free protein synthesis system. Isolation of RNA from subsynaptosomal components indicated that most, if not all, of the synaptosomal messenger activity was localized in the synaptic mitochondria.     

LOCALIZATION, SOLUBILIZATION AND PROPERTIES GANGLIOSIDE TRANSFERASES IN RAT BRAIN OF N-ACETYLGALACTOSAMINYL AND GALACTOSYL GANGLIOSIDE TRANSFERASES IN RAT BRAIN

Abstract— The subcellular distributions of UDP-N-acetylgalactosamine: G_M3 N-acetyl-galactosaminyl transferase and UDP-galactose: G_M2 galactosyl transferase, two enzymes involved in the biosynthesis of gangliosides, were determined in the 7-day-old rat brain by means of synaptosomal fractionation techniques. The enzymes were located on the synaptic membranes and appeared to be closely associated with gangliosides and acetylcholinesterase. Solubilization of the transferase enzymes from the microsomal particles was achieved and differed from the solubilization of acetylcholinesterase and of the total membrane protein. Competition studies suggest that the ^N-acetylgalactosaminyl transferase involved in the formation of G_M2 from G_M3 is different from the N-acetylgalactosaminyl transferase involved in the formation of GalNAoGal-Glc-ceramide from Gal-Glc-ceramide, whereas in contrast, both the formation of G_M1 from G_M2 and of Gal-GalNAc-Gal-Glcceramide from GalNAc-Gal-Glc-ceramide appear to be catalysed by the same galactosyl transferase.     

BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF ALKALOID-BINDING PROTEINS FROM IMMATURE RAT BRAIN1

Abstract— Vinblastine- and colchicine-binding proteins in the soluble fraction of immature rat brains were characterized and compared. Based upon criteria of Sephadex G-200 chromatography, electrofocusing and immunological reactivity, several separable species of vinblastine-binding protein were isolated. By contrast, these same procedures yielded only one protein band or elution peak to which [¹⁴C]colchicine could be tightly bound. This colchicine-binding protein peak coincided, in part, with one of the protein peaks to which [³H]vinblastine was tightly bound. Rabbit antiserum against soluble brain proteins precipitated by vinblastine sulfate contained antibodies which reacted with colchicine-binding protein. Thus, despite apparent differences in physical properties between the bulk of the vinblastine-binding proteins and the colchicine-binding protein, the vinblastine sulfate-precipitated protein antigens gave rise to antibodies capable of forming an immune complex with colchicine-binding protein.     

SOME IMMUNOCHEMICAL CHARACTERISTICS OF W1 AND W2 WOLFGRAM PROTEINS ISOLATED FROM RAT BRAIN MYELIN

Starting from a chloroform-methanol (2: 1 v/v) insoluble pellet of rat brain myelin, two pure proteins W1 and W2 were isolated by sodium dodecylsulphate preparative polyacrylamide gel electrophoresis. Their amino acid composition was compared. Antibodies against these proteins were prepared in rabbits. It was found that the two antigens have common antigenic similarities. The presence of one precipitin line of identity when myelin or isolated W1 and W2 from different animals were tested, led to the conclusion that there was no species specificity. The importance of the availability of such antisera is discussed.     

DEVELOPMENTAL PROFILES OF GANGLIOSIDES IN HUMAN AND RAT BRAIN

Abstract— The developmental profiles of individual gangliosides of human brain were compared with those of rat brain. Interest was focused mainly on the pre- and early postnatal development. Human frontal lobe cortex covering the period from 10 foetal weeks to adult age and the cerebrum of rat from birth to 21 days were analysed. Lipid-NANA and lipid-P were followed; in the rat, also protein and brain weight. A limited number of samples of human cerebral white matter and cerebellar cortex were also studied. The following major results were obtained:

• 1 The ganglioside concentration increased approximately three-fold within a short period: in rat cerebrum, from birth to the 17th day; in human cerebral cortex, from the 15th foetal week to the age of about 6 months. The largest increase in the rat brain occurred by the 11th to the 13th day; in human brain by term. The relative increase of gangliosides during this period was more rapid than that of phospholipids.
• 2 A hitherto unknown distinct early period of ganglioside and phospholipid formation in rat occurred by the second to fourth day.
• 3 The changes in brain ganglioside pattern, characteristic of the developmental stages of the rat, were found to be equally pronounced in the human brain.
• 4 Regional developmental differences in the ganglioside pattern were demonstrated in human brain. A characteristic white matter pattern, rich in monosialogangliosides, had developed by the age of 1 year. The increase in ganglioside concentration and the formation of the definitive ganglioside pattern of cerebellar cortex occurred later than in cerebral cortex. This cerebellar pattern was characterized by a very large trisialoganglioside fraction.
• 5 The two periods of rapid ganglioside metabolism in rat brain preceded the two periods of rapid protein biosynthesis.

     

ACETYLCHOLINE ESTERASE ACTIVITY OF SYNAPTIC VESICLE FRACTIONS AND MEMBRANE FRACTIONS PREPARED FROM RAT BRAIN TISSUE1

Abstract— —The synaptic vesicle fraction, M₂, isolated from rat brain tissue contained acetylcholine, acetylcholine esterase activity, and gangliosides. The separation of M₂ into several subfractions showed that the enriched synaptic vesicle subfraction, M₂A, contained acetylcholine, but little acetylcholine esterase and ganglioside. The membrane subfraction, M₂B, was enriched in the enzyme and ganglioside. Calcium ions facilitated the removal of membrane material from the synaptic vesicle fraction. In addition, the acetylcholine esterase of the synaptic vesicle fractions, M₂ and M₂A, frequently were activated by calcium ions as well as other cations.