INHIBITION OF RAT BRAIN PHENOL SULPHOTRANSFERASE IN VITRO BY NORADRENALINE and DOPAMINE METABOLITES1

Abstract— Kinetic parameters of the sulphotransferase reaction in rat brain were investigated in vitro at pH 7.4. Evidence is presented that the enzyme phenol sulphotransferase (EC 2.8.2.1) can be assayed with 4-methylumbelliferone or 3-methoxy-4-hydroxyphenylethyleneglycol as the substrate. Both assays give identical V_max values, whereas K_m values are 0.026 mm and 0.039 mm , respectively. Normetanephrine, metanephrine and the catecholamines adrenaline and dopamine, having a positive charge on the side chain at pH 7.4, do not inhibit 4-methylumbelliferone and 3-methoxy-4-hydroxyphenylethy-leneglycol sulphotransferase at this pH. Their deaminated metabolites 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxy-4-hydroxyphenethanol and 3-methoxy-4-hydroxyphenylacetic acid inhibit both the enzyme activities. The type of inhibition is noncompetitive with the exception of 3-methoxy-4-hydroxy-phenylethyleneglycol, which is a competitive inhibitor of 4-methylumbelliferone sulphation. 3-Methoxy-4-hydroxy-mandelic acid does not inhibit the enzyme activities. It is concluded that the catecholamines themselves are not sulphated by rat brain in vitro at pH 7.4.     

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.     

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。     

OCCURRENCE OF A SEROTONIN SULPHOTRANSFERASE IN THE BRAIN

Abstract— —An enzyme catalysing the transfer of sulphate from 3'-phosphoadenylsulphate to serotonin was purified from rabbit brain. The purification procedure involved ammonium sulphate fractionation of the 200,000 g supernatant of rabbit brain homogenate, treatment with alumina Cγ, and chromatography on DEAE-cellulose. The enzyme was purified 67-fold from the 200,000 g supernatant of the brain homogenate. The intracranial distribution of the sulphotransferase was investigated and the cerebellum found to have rather high activity. The sulphotransferase activities of rabbit, dog, rat and bovine brains were compared; rabbit brain had the highest activity, followed by dog, rat and bovine brain.     

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.     

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.     

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。     

INTRACELLULAR LOCALIZATION OF NATIVE AUXIN IN AVENA COLEOPTILE

Intracellular localization of the native auxin in the Avenacole-optile tip was investigated by separating cellular fractionsby differential centrifugation. Each fraction was extractedwith ether and the auxin activity was measured by the sensitizedAvena curvature test. After the removal of the native free auxin,each fraction was alkaline-hydrolyied, and from these hydrolyzatesthe bound auxin was extracted with ether and its activity wasmeasured. Both the native free auxin and the native bound auxinin these extracts were identified as IAA by paper chromatography.The results show that the native free auxin occurs only in thesupernatant soluble cytoplasm, and that the native bound auxinlocalizes also in the supernatant. The distribution of the externallyapplied IAA was also investigated. (Received February 27, 1962; )     

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.     

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.     

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.     

INTRACELLULAR LOCALIZATION OF SAXITOXINS IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The potent neurotoxin saxitoxin, and possibly several of its derivatives, are localized in two types of sites within the marine dinoflagellate Gonyaulax tamarensis Lebour. Immunocytochemical techniques using a polyclonal antibody and epifluorescence microscopy demonstrate toxin localization within the nucleus as well as on the periphery of small granules thought to be starch grains. In the nuclear region, the labelling occurred on or close to the permanently-condensed chromosomes as well as in an area within the two arms of the nucleus in the vicinity of the nucleous. No binding was observed in a closely-related, non-toxic dinoflagellate. Different binding affinities were observed between the nucleus and the grains at high and low antibody dilutions. This may relate to the polyclonal nature of the antiserum and to the presence of multiple toxins within the G. tamarensis isolate studied. Mechanistic interpretations of these labelling patterns remain speculative, especially the localization of the antigen at the outer edge of starch grains, but the distinct labelling in the nuclear region suggests that saxitoxin, with its two positively charged guanidinium groups, may bind to nucleic acids or nuclear proteins in a manner analogous to the polyamines and other cations. The labelling patterns reported here suggest that the saxitoxins may not simply be secondary metabolites but instead could be important compounds involved in the structure and function of the G. tamarensis genome.     

BIOGENIC AMINES AND THEIR METABOLITES AS SUBSTRATES FOR PHENOL SULPHOTRANSFERASE (EC 2.8.2.1) OF BRAIN AND LIVER

Phenol sulphotransferase activity in homogenates of rat liver and brain was determined spectrophotometrically. Rat liver had about 100-fold more phenol sulphotransferase activity than brain; however, both tissues showed about the same spectrum of activity towards the phenolic compounds tested. Dopamine and its acidic and neutral metabolites and the neutral metabolites of norepinephrine were the compounds most readily sulphury-lated in vitro. They were also the compounds most readily sulphurylated in vivo when they were injected intraventricularly together with labelled Na₂SO₄. When labelled Na₂SO₄ was injected alone, we detected conjugation of endogenous phenols. One of the compounds formed was identified by its chromatographic characteristics as 3-methoxy-4-hydroxy-phenylethyleneglycol sulphate. We detected other conjugates which appeared to be the sulphate esters of 3,4-dihydroxyphenylethyleneglycol; 3,4-dihydroxyphenylacetic acid; and homovanillic acid. In brain, sulphate conjugation may be a major route of metabolism for many of the phenolic compounds related to the biogenic amines and possibly for phenolic drugs which enter the brain.     

INTRACELLULAR DISTRIBUTION OF ALKALINE PHOSPHATASE IN RAT LIVER CELLS

1. Cytochemical studies of the intracellular distribution of alkaline phosphatase in rat liver have been made, using a fractionation procedure recently developed in this laboratory (8) and a similar but modified method not described previously. Aqueous media were used in both cases. 2. The alkaline phosphatase was found to consist of two forms, one of which is strongly activated by magnesium and one of which is not sensitive to this metal. 3. The form of the enzyme that is not activated by magnesium occurs mainly in the nuclear fraction, where it seems to be rather firmly bound. Some of this form of the enzyme is also found in the microsomes, but very little if any occurs in the soluble supernatant fraction. 4. The form of alkaline phosphatase which is activated by magnesium occurs mainly in the soluble supernatant fraction, but what is believed are significant amounts also occur in nuclei. A significant portion of this form of the enzyme can be extracted from the isolated nuclei with cold, isotonic saline solution. Some activity of this form of the enzyme is also found in the microsomal fraction. 5. Mitochondria appear to contain relatively little alkaline phosphatase of either kind. 6. The concept of a porous nuclear membrane has been invoked to explain some of the results obtained in this work. It is postulated that part at least of the form of the enzyme that is activated by magnesium is free to diffuse back and forth through pores in the nuclear membrane, whereas this is considered not to be possible for the form of the enzyme that is insensitive to magnesium as a result of the firm binding of the latter to nuclear substance.     

LOCALIZATION OF THE THY-1 ANTIGEN IN THE CEREBELLAR CORTEX OF RAT BRAIN BY IMMUNOFLUORESCENCE DURING POSTNATAL DEVELOPMENT

Abstract— At birth in the rat brain the Thy-1 antigen was present at 10% of the adult level and increased rapidly to reach near adult levels after 3 weeks. Localization studies by immunofluorescence on sections of rat cerebellar cortex during this period showed that at day 5 there was weak fluorescence associated mainly with the molecular layer and some fibre-like structures in the centre of the folium; no fluorescence was found around the cells of external granular layer. From 5 to 16 days there was a rapid increase in Thy-1 immunofluorescence with noticeably higher levels associated with the white matter than the molecular layer. However, by 21 days the reverse was found' with lower levels in white matter than in the molecular layer with a similar distribution to that observed previously in adult rat cerebellum. Small rings and patches of fluorescence were observed in the molecular and granular layers. The results indicated that Thy-1 was present on axons, mature neurons and their processes. In addition, Thy-1 immunofluorescence was found in the pia-arachnoid until around day 16.