TOPOGRAPHICAL AND SUBCELLULAR DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE IN PIGEON OPTIC TECTUM

Abstract— The distribution of choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in different layers of the pigeon optic tectum and in some nuclei of the optic lobe have been investigated. About 40% of GAD and 25% of ChAT were found in the superficial part of tectum, but negligible activity was found in the stratum opticum. The highest GAD activity was found in layers 3-7 (according to the nomenclature of C ajal , 1911) with a peak in layer 4. ChAT activity peaked in layers 3, 5. 8 and 10/11. Its distribution correlated well with the staining pattern of AChE, particularly in the superficial part of the tectum. The distribution of ChAT and GAD did not change significantly 4 weeks after enucleation. ChAT and GAD activities were high in the nucleus isthmi, pars parvocellularis (Ipc). The activity of GAD was also high in the nucleus intercollicularis (ICo), the other nuclei showed less activity of both enzymes.     

MEMBRANE AFFINITIES AND SUBCELLULAR DISTRIBUTION OF THE DIFFERENT MOLECULAR FORMS OF CHOLINE ACETYLTRANSFERASE FROM RAT

Choline acetyltransferase from rat brain is present in three different molecular forms with isoelectric points at pH 7·4-7.6, 7·7-7·9 and 8·3. The three forms were identified in a highly purified enzyme preparation, in a preparation of synaptosomes and in a cyto-plasmic preparation from disrupted axons and perikarya (fraction S₃). The three molecular forms differed in their affinities for synaptosome membranes and for a cation exchange resin (CM-Sephadex C-50). The positive surface charges of the different molecular forms and their affinities for membranes correlated well with their isoelectric points. The molecular form with jsoelectric point 8·3 had the largest positive surface charge and the highest membrane affinity. On isoelectric focusing of an extract from rat brain synaptosomes, the molecular form with isoelectric point 8·3 formed a complex with a negatively charged compound, presumably a protein. A method was developed to remove this compound by treatment with DEAE-Sephadex or by precipitation with vinblastine. These procedures are similar to methods known to remove the neurotubular protein. The complex formation did not occur in fraction S₃.     

THE DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN VERTEBRATE RETINA1

Abstract— Choline acetyltransferase (ChAc) activity was determined in retinal layers from 10 vertebrates. In all animals, the highest activity was in the inner plexiform layer, intermediate activity in the inner nuclear and ganglion cell layers, and very low activity in the photoreceptor and outer plexiform layers and optic nerve. The pattern of distribution of enzyme activity within the inner nuclear layer corresponds quantitatively to the distribution of amacrine cells within that layer. A species difference of almost 90-fold was found between the lowest and highest values for ChAc activity in inner plexiform layer. The variation in enzyme activity found among homeotherms in inner nuclear and inner plexiform layers is related to the number of amacrine cell synapses in the inner plexiform layer. But the differences in enzyme activity are generally greater than those which have been found in numbers of amacrine cell synapses between species. The data suggest that cholinergic neurons in retina are to be found predominantly among the amacrine cell types and that not all amacrine cells will be found to be cholinergic.     

THE SUBCELLULAR DISTRIBUTION OF PARTICLE-BOUND NEGATIVE CHARGES IN RAT BRAIN

Abstract— Ferritin cationized with Girard's reagent T [(CH₃)₃NCH₂CONHNH₂] was used as a marker for negative electric charges on subcellular components of rat brain. Pre- and postsynaptic membranes did not bind cationic ferritin, either on their outer or their inner surface. Vesicular membranes, however, were heavily labelled with cationic ferritin. Post- and presynaptic densities appeared to carry a large number of negative charges. Cationic ferritin was also bound within the synaptic cleft. The density varied, however, from synapse to synapse: in general, binding within the cleft was significantly reduced when the synaptosomes were pretreated with 1 M-NaCl. Cationic ferritin was observed in high density on the outer surface of myelin sheaths and even between the lamellae at the intraperiod lines. Neuro-tubules and neurofilaments bound cationic ferritin in significant amounts. The results are discussed in relation to the mechanism of neurotransmission and to the binding and transport of drugs.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

TOPOGRAPHICAL AND SUBCELLULAR LOCALIZATION OF CHOLINE ACETYLTRANSFERASE IN RAT HIPPOCAMPAL REGION

—Choline acetyltransferase (ChAc) was localized in discrete layers in hippocampus regio superior and in area dentata. The highest activity in hippocampus was found in a narrow infrapyramidal zone, but high activities were also observed in the rest of stratum oriens and in stratum pyramidale. In area dentata the highest activities were found in a narrow supragranular zone and in hilus fasciae dentatae. The localization corresponded very closely to that of acetylcholinesterase. The main part of ChAc activity was confined to the synaptosome fraction. The results are compatible with the view that pyramidal and granular cells are excited by cholinergic boutons, mainly located on the basal or apical dendrites near the somata.     

THE SUBCELLULAR DISTRIBUTION OF CARBONIC ANHYDRASE IN HOMOGENATES OF PERFUSED RAT BRAIN

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.
Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.
Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten-fold increase in the measurable carbonic anhydrase activity of myelin fragments.     

DIFFERENCES IN THE OCCURRENCE AND DISTRIBUTION OF HYDROXYPROLINE-PROTEINS AMONG THE ALGAE

Fifty algae from seven phyla have been examined in order to determine whether they contain protein-bound hydroxyproline and whether this hydroxyproline is concentrated in the cell wall. Green algae, with the exception of Nitella, all contain hydroxyproline, and in most cases it is concentrated in the cell wall. Hydroxyproline is also present in low levels in the brown algae, but here it is concentrated in the soluble proteins. Red algae contain no hydroxyproline. The presence of hydroxyproline in blue-green algae is variable, but when present the levels are low. It appears, then, that the major algal phyla differ with respect to the distribution and occurrence of hydroxyproline-proteins.     

THE REGIONAL DISTRIBUTION, AGE DEPENDENT VARIATION AND SPECIES DIFFERENCES OF BRAIN ARYLSULPHATASES

Abstract— The relative proportions of arylsulphatase A and B were determined by the method of B aum , D odgson and S pencer (1959) in brains of various animal species and it was found that there was a considerable variation in the concentration of these two enzymes.
Arylsulphatase A and B of various animal species including rat, man, monkey, sheep and chicken were partially separated using zinc acetate fractionation procedure and gel electrophoresis. The chicken brain arylsulphatase A had a similar electrophoretic mobility to that of arylsulphatase B of other species. Further, chicken brain arylsulphatase A precipitated at a zinc acetate concentration of 0005 M, a condition under which arylsulphatase B from the brain of other species precipitated.
Kinetic properties such as K _m value and inhibitory effect of sulphite and phosphate ions indicated that chicken brain arylsulphatase A was similar to arylsulphatase A of other species.
The results on regional distribution of arylsulphatase A and B activities in monkey brain and in developing rat brain suggest a relationship between arylsulphatase A and sulphatides and arylsulphatase B and mucopolysaccharides.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

SUBCELLULAR DISTRIBUTION OF CEREBRAL CHOLE-STANOL IN CEREBROTENDINOUS XANTHOMATOSIS

Abstract— Frozen frontal lobe from a patient with cerebrotendinous xanthomatosis and two control specimens were separated by differential centrifugation into subcellular fractions. The fractions were differentiated by their electron microscopic appearance, by their succinate dehydrogenase and acetylcholinesterase activities and by their galactolipid contents. Free cholestanol was present in largest amounts in the myelin fraction and was also found in each subcellular fraction prepared from the cerebrotendinous xanthomatosis brain. Thus, in this condition, cholestanol storage is a property of myelin and probably also of other brain membranes.     

SUBCELLULAR DISTRIBUTION AND SOME PROPERTIES OF ACETYL-COENZYME A HYDROLASE IN THE BRAIN

—The detailed subcellular distribution and some properties of acetyl-CoA hydrolase were studied in the rat brain. The brain homogenate (S₁) hydrolysed acetyl-CoA at a rate of approx 2·3 nmol/min/mg of protein at 37°C. The total activity of acetyl-CoA hydrolase was distributed in the following order: soluble > mitochondrial > microsomal, synaptosomal > myelin fraction. The order of the specific activity of the enzyme was: soluble, microsomal > mitochondrial > synaptosomal > myelin fraction. The synaptic vesicle fraction (D) had relatively high specific activity among the intraterminal particulate fractions, having two or three times higher specific activity than that of the synaptic cytoplasmic membrane fraction (F or G). Attempts to de-occlude acetyl-CoA hydrolase in the particulate fraction showed that only the enzyme activity in the myelin fraction was increased markedly by the treatment with ether or Triton X-100. Lineweaver-Burk plots gave straight lines for each subcellular fraction and apparent K_m values for acetyl-CoA were between 0·1 and 0·2 mM. Neither diisopropyl fluorophosphate nor physostigmine at the concentration of 0·1 mm inhibited the enzyme activity.