IDENTIFICATION OF MULTIPLE FORMS OF AMINOBUTYRATE TRANSAMINASE IN MOUSE AND RAT BRAIN : SUBCELLULAR LOCALIZATION

—(1) At least four distinct molecular forms of 4-aminobutyrate: 2-oxoglutarate aminotransferase from mouse and rat brain, have been separated by electrophoresis on paper, cellogel, agargel, silicagel and by immunoelectrophoresis. (2) The existence of specific typical electrophoretic profiles in mitochondrial and extramitochondrial compartments was shown. (3) A differential effect of pH on the anionic and cationic 4-aminobutyrate:2-oxoglutarate aminotransferase transaminase activities has been shown. (4) The possible consequences of the 4-aminobutyrate: 2-oxoglutarate aminotransferase isozyme compartimentation on the local availability of γ-aminobutyric acid pools has been discussed.     

APPEARANCE OF GAMMA AMINOBUTYRATE TRANSAMINASE ACTIVITY IN DEVELOPING RAT BRAIN

—The distribution, localization and changes in intensity of γ-aminobutyrate transaminase (4-aminobutyrate: 2-oxoglutarate aminotransferase, EC 2.6.1.19) in rat brain have been studied during the first 20 days of postnatal life by a histochemical technique. Enzyme activity at birth was seen only in Purkinje cells of the cerebellar cortex where it increased markedly during the first 20 days. A rapid increase in enzyme activity was seen in regions of the hind-brain after 3 days but a slower increase was apparent in areas of the fore- and mid-brain. The results indicated that by 10 days post-partum nerve cell GABA-T activity had developed in the majority of brain areas studied, while glial cell GABA-T activity developed between 10 and 15 days post-partum. Evidence is presented which indicates that there is a discontinuous function of GABA-T in the developing brain.     

INTRACELLULAR LOCALIZATION OF PHENOL SULPHOTRANSFERASE IN RAT BRAIN

—The intracellular localization of phenol sulphotransferase in rat brain was studied The distribution pattern found after differential centrifugation closely resembles that of lactate dehydrogenase and does not change during postnatal development. The distribution of the enzyme in discontinuous and continuous sucrose gradients, however, shows a deviation from the lactate dehydrogenase pattern and a shift towards a higher sucrose concentration during development. In the adult the phenol sulphotransferase coincides with monoamine oxidase, succinate dehydrogenase and β-glucuronidase. Disruption experiments, purification of mitochondria and electron microscopy exclude localization of phenol sulphotransferase in mitochondria. These studies support the idea of phenol sulphotransferase as a cytoplasmic enzyme with a preferential binding to or localization in oligodendroglial cells or, more probably, a specific type of synaptosomes.     

THE LOCALIZATION OF ENZYME ACTIVITIES IN THE RAT BRAIN

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.¹ Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature.     

SEMIAUTOMATED FLUORIMETRIC DETERMINATION OF TYROSINE IN RAT BRAIN AND HUMAN PLASMA

Abstract— The evidence is presented to show that the fiuorimetric method of W aalkes & U denfriend (1957) cannot be used directly for the determination of free tyrosine in acid deproteinized extracts of rat brain, owing to the reaction of peptide bound tyrosine in some proteins present in the acid extracts. A method is given to obtain a fraction containing free tyrosine in the acid extracts of rat brain and to determine its tyrosine content fluorimetrically by autoanalysis. An autoanalysis method is also given for the fiuorimetric determination of free tyrosine directly in 5₀ trichloroacetic acid extracts of the plasma.     

TYROSINE HYDROXYLASE IN RAT BRAIN: DEVELOPMENTAL CHARACTERISTICS

Abstract— The development of tyrosine hydroxylase (tyrosine 3-hydroxylase, EC 1.14.3.a) activity has been examined in whole rat brain and in various regions and subcellular fractions thereof. The specific activity of tyrosine hydroxylase increased almost 15-fold from 15 days of gestation to adulthood. With maturation, those regions of the brain that contain only terminals of the catecholaminergic neurons showed the greatest increases in enzyme activity. There was a shift in the subcellular distribution of tyrosine hydroxylase from the soluble fraction in the fetal brain to the synaptosomal fraction in the adult brain. Tyrosine hydroxylase, dopamine hydroxylase (EC 1.14.2.1) and the specific uptake mechanism for norepinephrine appear to develop in a coordinated fashion.     

雌激素Beta受体在大鼠脑内表达的免疫组化定位研究

为了探讨雌激素作用于神经系统的机理,采用硫酸镍铵增强显色的免疫组化SP法研究了新的雌激素受体(ER-β)在成年雌雄大鼠脑内的分布。研究证实ER-β免疫阳性物质主要位于神经元的细胞核内,但在个别脑区也可在胞浆甚至突起内检测到。最强的ER-β免疫阳性信号见于前嗅核、大脑皮质、小脑浦肯野细胞、斜角带垂直部、蓝斑和三叉神经运动核等部位;中等强度的染色见于隔内侧核、杏仁外侧核、黑质、中央灰质等部位;较弱的阳性反应见于下丘脑与杏仁复合体的部分核团。在一些部位还存在表达水平甚至细胞内定位模式的性别差异,如前庭上核内的表达只见于雌性;雄性大鼠三叉神经运动核内ER-β蛋白主要表达于胞浆内,细胞核为阴性;而在雌性大鼠该部位ER-β蛋白主要位于细胞核等。以上结果表明ER-β蛋白在大鼠脑内分布广泛并具有一定的性别差异,在与学习记忆有关的脑区如大脑皮质和基底前脑内有很高的表达,提示在脑组织内雌激素可能通过ER-β这一新的信号途径发挥多种重要的调控作用,如学习记忆等。     

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.     

THE SUBCELLULAR LOCALIZATION OF HISTIDINE DECARBOXYLASE IN VARIOUS REGIONS OF RAT BRAIN

Abstract— The subcellular distribution of histidine decarboxylase (assayed by two different isotopic methods) and several biochemical markers (lactate dehydrogenase, DOPA decarboxylase and protein) was determined in rat cerebral cortex. After differential centrifugation, the enzyme activity was found mainly in the crude mitochondrial and soluble fractions. Further separation of the former on discontinuous sucrose gradients showed that the particulate histidine decarboxylase (HD) was found in the synaptosomal fraction. After osmotic shock, HD activity appeared in the supernatant fraction suggesting that a major portion of the enzyme is localized in the cytoplasm of cortical nerve endings. By analogy with other brain amines, this finding, together with the presence of histamine in synaptic vesicles (K ataoka and de R obertis , 1967), can be taken as further support for the hypothesis of a role as neurotransmitter for histamine.
Various brain regions were homogenized under conditions leading to synaptosome formation. The distribution of HD between 'particulate' and soluble fractions differed from one region to the other, but did not give any clear-cut indication of regions rich in cell bodies or nerve terminals.     

谷氨酸转运体GLAST mRNA在大鼠脑内表达的细胞定位

实验采用荧光双标技术研究谷氨酸转运体ＧＬＡＳＴ ｍ ＲＮＡ 在大鼠脑内表达的细胞定位, 研究表明, 在星形神经胶质细胞和神经元, ＧＬＡＳＴｍ ＲＮＡ 分别与神经胶质纤维蛋白（ＧＦＡＰ） 和神经元特异性烯醇化酶 （ＮＳＥ） 有表达共存, 提示ＧＬＡＳＴ ｍ ＲＮＡ在星形神经胶质细胞和神经元上都有表达。     

实验性运动病大鼠神经细胞钙离子超微结构定位变化

本研究采用正负交变加速度旋转刺激法制备大鼠运动病模型,并用钙离子（Ｃａ２＋）超微结构定位法观察了运动病大鼠大脑皮质、小脑皮质和脑干前庭区神经细胞中的Ｃａ２＋变化。结果表明,运动病大鼠大脑皮质、小脑皮质和脑干前庭区神经细胞胞质基质、线粒体和内质网中Ｃａ２＋反应产物增多。提示运动病的发生与中枢神经细胞Ｃａ２＋内流有关。     

BRAIN TYROSINE HYDROXYLATION: ALTERATION OF OXYGEN AFFINITY IN VIVO BY IMMOBILIZATION OR ELECTROSHOCK IN THE RAT

Abstract— The rates of brain tyrosine and tryptophan hydroxylation, estimated in vivo from the accumulation of DOPA and 5-hydroxytryptophan after the administration of a decarboxylase inhibitor, appear dependent on the availability of oxygen as a substrate. During two types of physical stress, electroshock and curare-immobilization, the rate of brain tyrosine hydroxylation was greater than in unstressed controls and was not significantly decreased when the stresssed animals were made hypoxic. The loss of oxygen dependence by brain tyrosine hydroxylation during stress was observed in several brain regions and was not associated with alterations in the concentrations of brain tyrosine. tryptophan, serotonin, dopamine or norepinephrine. The rate of brain tryptophan hydroxylation was not affected by stress and remained oxygen dependent. The increase in catecholamine synthesis during stress appears to be the result of increased catecholaminergic nerve impulse flow. These experiments are consistent with the hypothesis that during neuronal stimulation an allosteric change in tyrosine hydroxylase increases the affinity of the enzyme for oxygen allowing greater catecholamine synthesis despite limiting concentrations of this substrate.     

ANALYSIS OF THE MAJOR AMINO ACIDS OF RAT BRAIN AFTER IN VIVO INHIBITION OF GABA TRANSAMINASE BY ETHANOLAMINE O-SULPHATE

The effect of in vivo inhibition of GABA transaminase by ethanolamine O-sulphate on the content of the free amino acids in rat brain has been studied. Intracisternal injection of 2.0 mg/kg resulted in a progressive increase in GABA levels with time, to reach after 8 h a 100 per cent increase over saline-injected control animals. The effect of injection of 0.5, 1.0 and 2.0 mg/kg was studied 24 h after injection and the results showed that the increased GABA levels were dependent on the dose of inhibitor employed. Apart from the substantial increase in the GABA concentration of the brain there were no significant changes in the content of the other amino acids except for a small but significant decrease in aspartic acid in one experiment. When the extent of inhibition of the transaminase was correlated with the rise in GABA concentration it was shown that no elevation occurred until more than half of the enzymic activity had been inhibited.     

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.     

SIALYLTRANSFERASES IN RAT BRAIN: INTRACELLULAR LOCALIZATION AND SOME MEMBRANE PROPERTIES

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.
The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM_1a, 700; exogenous DS-fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza-tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.     

ASSAY AND PROPERTIES OF 4-AMINOBUTYRIC-2-OXOGLUTARIC ACID TRANSAMINASE AND SUCCINIC SEMIALDEHYDE DEHYDROGENASE IN RAT BRAIN TISSUE

Abstract— The activity of 4-aminobutyric-2-oxoglutaric acid transaminase (GABA transaminase) and succinic semialdehyde dehydrogenase was determined in total rat brain homogenate. GABA transaminase activity was measured using a coupled enzyme method which utilizes endogenous succinic semialdehyde dehydrogenase to convert the formed succinic semialdehyde into succinate. The concurrently produced NADH was used as an estimate of GABA transaminase activity. This method could be used since it was shown that the dehydrogenase was about twice as active as the transaminase and because no significant accumulation of the intermediate succinic semialdehyde could be detected. GABA transaminase was inhibited by high ionic strength. In contrast NaCl decreased the apparent K _m and increased V _max for succinic semialdehyde dehydrogenase at high but not al low tissue concentrations. Increasing tissue concentration also resulted in a decrease of the apparent K _m, but did not change the V_max of succinic semialdehyde dehydrogenase and it is suggested that this enzyme can exist in two distinct states of aggregation, one with a high and one with a low affinity for succinic semialdehyde. The high affinity form of the enzyme is thought to prevent succinic semialdehyde from accumulation in the GABA transaminase assay. It is concluded that within certain limits the coupled enzyme method described here can be used for the assay of GABA transaminase activity.     

LOCALIZATION OF GLUTAMINASE IN THE RAT NEOSTRIATUM

Abstract— Glutaminase activity in rat striatal tissue was not significantly decreased by lesions of the cortico-striatal tract which depressed striatal glutamate uptake by 47% but was markedly decreased following intrastriatal injections of kainic acid. There also appeared to be a linear correlation between glutaminase and glutamic acid decarboxylase activities in the substantia nigra of rats injected intrastriatally with kainic acid. The results suggest that most of the glutaminase activity in these regions is localized in GABAergic structures and provide no evidence for the occurrence of this enzyme in nerve endings of the glutamergic cortico-striatal tract.     

DIFFERENCES IN THE REGULATION OF TYROSINE AMINOTRANSFERASE IN BRAIN AND LIVER

Abstract— l -Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) activity in rat brain is not regulated in the same way as in rat liver. No diurnal rhythm in the activity of the cerebral enzyme was found in rats fed ad lib. although there was a marked diurnal variation in the activity of the hepatic enzyme. In adrenalectomized rats, hydrocortisone and glucagon induced the enzyme in liver but had no effect on the enzyme in brain. In normal rats, treatment with reserpine or exposure to cold elevated the activity of the hepatic enzyme without affecting the enzyme in brain. Thus, the tyrosine aminotransferase of brain differed from the enzyme in liver since it did not exhibit diurnal variations of activity and was not affected by hormones, drugs, or stress.