ACTIVATION, INHIBITION AND AGGREGATION OF CHOLINE ACETYLTRANSFERASE (EC 2.3.1.6)

—Mercuric chloride, silver acetate and cupric sulphate (0·1 mm ) completely inhibited purified choline acetyltransferase from bovine caudate nuclei. At the same concentration cadmium chloride and zinc acetate gave a 50 per cent inhibition. Potassium and sodium salts more than doubled the enzymatic activity while creatinine hydrochloride more than tripled it. Guanidine hydrochloride was less effective than creatinine hydrochloride but more effective than KCl and NaCl. Sodium chloride and creatinine hydrochloride had a synergistic effect on the enzyme. When ammonium sulphate was used to fractionate the choline acetyltransferase that had been extracted from bovine caudate nuclei, the enzyme aggregated into different molecular sizes as determined by exclusion chromatography on Bio-gel A-1·5 m. The molecular weight of the largest aggregate was at least 10⁶ daltons. The initial tissue extract contained only one molecular species of ChAc as did a partially purified preparation in which ammonium sulphate was not used in the purification.     

PURIFICATION AND SOME PROPERTIES OF CHOLINE ACETYLTRANSFERASE (EC 2.3.1.6) FROM BOVINE BRAIN

Abstract— Choline acetyltransferase (ChAc) has been isolated and highly purified from the caudate nuclei of bovine brains. The procedure involved: (1) making an acetone- and chloroform-insoluble powder from the tissue; (2) treating the powder with aqueous buffer and chromatographing the extractable soluble proteins on an organomercurial-sepharose column; (3) removing impurities by passage through columns of DEAE-cellulose and hydroxyapatite; and (4) separation of the heme-containing protein from ChAc by denaturing the former with a mixture of chloroform and n -butanol. The purified ChAc was essentially homogeneous as judged by polyacrylamide gel electrophoresis and exhibited a pH optimum at about pH 7. The partially purified ChAc dissociated into two non-identical subunits when chromatographed with a dilute buffer on Bio-gel A. It did not dissociate when a more concentrated buffer was used. The purified ChAc dissociated on the Bio-gel A even in the presence of a high salt concentration. The dissociation was accompanied by a great loss of enzymatic activity, and we concluded that the presence of other proteins tends to prevent the dissociation of ChAc on gel filtration.     

KINETICS OF CHOLINE ACETYLTRANSFERASES (EC 2.3.1.6) FROM HUMAN AND OTHER MAMMALIAN CENTRAL AND PERIPHERAL NERVOUS TISSUES

Abstract— Choline acetyltransferase (EC 2.3.1.6) was partially purified from human caudate nucleus and putamen, human sciatic nerve, rabbit and rat brain, and rabbit sciatic nerve. Kinetic constants were determined under the same conditions for all six extracts. Extrapolated K_m values were between 6.6 and 18 μM for acetyl-CoA and between 0.4 and 1.2 mM for choline. Product inhibition patterns indicated that ChAc from both central and peripheral nervous tissues of man and the rabbit obeys a Theorell-Chance mechanism. Kinetic parameters suggest a possible influence on ACh synthesis of the in vivo concentration ratio, CoA/acetyl-CoA.     

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.     

INHIBITION OF CHOLINE ACETYLTRANSFERASE AND ITS HISTOCHEMICAL LOCALIZATION

Abstract— A method for the histochemical identification of choline acetyltransferase has been investigated further by studying the effects of certain inhibitors of the enzyme both on rat brain homogenates and on the localization of the enzyme in tissue sections.
It was confirmed that acetyl-CoA hydrolase activity both in homogenates and in tissue sections is inhibited by preincubation in 1 mM-DFP. The effects of the choline acetyltransferase inhibitors chloro- and bromoacetylcholine on the appearance of histochemical staining were related to their activity in homogenates and tissue slices. Bromoketone was found to inhibit choline acetyltransferase in homogenates and, less efficiently, in tissue sections but it also inhibited the hydrolysis of acetyl-CoA by some other unknown enzyme which is inactivated by 1 mM-DFP.
The results obtained with the choline acetyltransferase inhibitors provide support for the specificity of the histochemical method.     

PURIFICATION OF CHOLINE ACETYLTRANSFERASE FROM DROSOPHILA MELANOGASTER

Abstract— Purification of choline acetyltransferase (ChAc) from heads of Drosophila melanogaster , the richest known source of ChAc, has been accomplished. The stability of the enzyme was preserved by working with a concentration of protein above 0.1 mg/ml. The purification was carried out with ammonium sulfate fractionation and column chromatography on QAE-Sephadex, CoA-Sepharose, G-200 Sephadex, and PCMB-Sepharose. In a procedure using 100 g of Drosophila heads, the specific activity of the crude homogenate was 0.028 μmol/min/mg protein and that of the final product was 43 μmol/min/mg protein, representing a 1500 fold purification. A single protein band, containing all of the ChAc activity, was seen by polyacrylamide gel electrophoresis. A sharp pH optimum at 7.2 was observed. Apparent K_m's for acetyl CoA and choline were 90 μM and 47 μM , respectively. The molecular weight was determined to be 69,000. Isoelectric focusing of extracts of Drosophila heads showed only one peak of choline acetyltransferase activity with an apparent pi of 5.1.     

N-SUBSTITUTED CHOLINE ANALOGUES AS SUBSTRATES FOR CHOLINE ACETYLTRANSFERASE

Abstract— Alkyl, phenyl, phenylalkyl and pyridiniumalkyl derivatives of choline were studied as substrates for choline acetyltransferase from bovine brain. When one methyl group of choline was replaced by an ethyl group, pK _m (negative logarithm of apparent K _m) decreased, whereas V _max was not significantly changed in comparison with choline. The n-propyl derivative showed an even lower pK _m with unchanged V _max Further elongation of the n-alkyl chain had little effect on the substrate parameters until the n-decyl derivative was reached, when a pronounced decrease of V _max occurred. The highest n-alkyl homologue studied, n-pentadecyl choline, was a very poor substrate. Phenylcholine was also a poor substrate, but introduction of an alkyl chain between the phenyl group and the quaternary nitrogen resulted in compounds with better substrate properties, although they were still inferior to choline. The lowest homologue of the pyridiniumalkyl cholines studied, pyridiniumpropylcholine, had a very low pK _m and a lower V _m in comparison with choline. Increasing the chain length of the alkyl residue resulted in an increase of pK _m, whereas V _max was little affected. The results demonstrate that replacement of one methyl group of choline with a more bulky substituent resulted in impaired substrate properties, presumably due to steric effects. No evidence was obtained for hydrophobic interactions between the enzyme and non-polar substituents in the choline analogues studied.     

SURFACE CHARGE OF CHOLINE ACETYLTRANSFERASE FROM DIFFERENT SPECIES

—The adsorption of partially purified choline acetyltransferase (ChAc) from cat, rat, guinea-pig and pigeon brains by the cation exchange resins, CM-Sephadex (C-50) and Amberlite CG-50 II, was studied at various pH values and ionic strengths. ChAc from cat and rat were more strongly adsorbed by cation exchangers and therefore have a stronger net positive surface charge than those from guinea pig and pigeon. Experiments showed that the difference in adsorption between these two groups of enzymes could not be explained by overloading of the resin, by competitive effect of other proteins present in the enzyme preparations or by the presence of any component suppressing the adsorption of ChAc in any of the enzyme preparations. The adsorption of ChAc by a cation exchanger is very similar to its binding to synaptosome membranes. The significance of the positive surface charge of ChAc in studies on the compartmentation of ChAc in synaptosomes is discussed.     

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.     

CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN CORPUS STRIATUM OF RESERPINISED RATS1

Rats treated with reserpine show increased V_max for the high affinity uptake of choline into small slices of corpus striatum. The choline acetyltransferase activity of whole homogenates of striatum is also increased. These changes are consistent with increased cholinergic neuronal activity in the striatum and seem likely to be adaptations mediating increased rates of synthesis of acetylcholine. The maximal increases found occurred concurrently, consistent with coupling of the high affinity uptake of choline and its acetylation in cholinergic nerve terminals of the rat. That increased high affinity uptake is accompanied by increased choline acetyltransferase activity, suggests the input of choline is not the sole determinant of rates of synthesis of acetylcholine, in spite of the large Vmas for striatal choline acetyltransferase, compared with that for high affinity uptake. These results seem best explained by kinetic coupling, in the rat, of the high affinity uptake of choline with a limited pool of choline acetyltransferase preferentially localised at the nerve terminal plasma membrane.     

CHOLINE ACETYLTRANSFERASE LEVELS IN DIENCEPHALIC NUCLEI OF THE RAT

Choline acetyltransferase levels have been measured in specific nuclei of the diencephalon. On the whole, the thalamic nuclei contain higher concentrations of this enzyme than do the hypothalamic and preoptic nuclei. Those nuclei which seem to receive the most dense cholinergic innervation contain the highest levels of choline acetyltransferase.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM TORPEDO CALIFORNICA

Abstract— Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine in nervous tissue, has been purified to apparent homogeneity from the electric organ of the electric fish Torpedo californica using ion-exchange, gel filtration, and hydroxyapatite chromatography. The final preparation had been purified 8570-fold to a specific activity of 30μmol ACh formed/min/mg protein. The purified protein has a pH optimum of 6.8 (phosphate buffer), is activated by low concentrations (ca. 10 m m ) of ammonium or alkylammonium ions, and is strongly inhibited by a sulfhydryl blocking reagent (DTNB). ChAT has a mol. wt. of 63000 when measured by SDS-polyacrylamide gel electrophoresis or gel filtration.
A new method for the rapid assay of ChAT activity is described in which unreacted substrate ([³H]acetyl-CoA) is removed from reaction mixtures by adsorption to charcoal: some advantages of this technique are discussed.     

PURIFICATION OF RAT BRAIN CHOLINE ACETYLTRANSFERASE AND AN ESTIMATION OF ITS HALF-LIFE

—Acetyl-CoA:choline-O-acetyltransferase (ChAc, EC 2.3.1.6) was purified from rat cerebral cortex and its half-life determined. The molecular weight of the enzyme under non-denaturing conditions was estimated by gel filtration to be in the range of 60,000–65,000. On SDS acrylamide gels, the purified enzyme migrated as a single band with a molecular weight estimated as 62,000. The turnover rate of ChAc in the mature rat was determined by the double label method, employing l -[1-¹⁴C]leucine and l -[4,5-³H]leucine. Its half-life under steady-state conditions was estimated to be 5.2 days. As a control, tubulin was isolated from the same preparation and its half-life measured. Under these conditions tubulin exhibited a half-life of 3.8 days.     

CHOLINE ACETYLTRANSFERASE ACTIVITY IN LARGE VENTRAL SPINAL NEURONS

Abstract— Up to approx 3 pmol of acetylcholine (ACh)/h/cell body was synthesized by perikarya of large spinal neurons isolated in bulk fractions from bovine ventral spinal cord. Many of the cell bodies are probably derived from motoneurons. A medium of low ionic strength and pH was used to minimize losses of soluble acetyl CoA:choline- o -acetyltransferase (ChAc; EC 2.3.1.6) from the neurons, whose permeability properties were altered. Such a medium also increased the retention of other soluble proteins by the cell bodies. The maximal rate of hydrolysis of ACh by the isolated neurons exceeded that of its synthesis by a factor of at least 100. It was estimated that ChAc and acetylcholinesterase (AChE; EC 3.1.1.7) each represent less than 0.01% by weight of the total protein in these cell bodies and that as little as 10% of each enzyme in the ventral spinal cord is located within the large neuronal somata and their proximal processes.     

CHOLINE ACETYLTRANSFERASE CONTENT IN DISCRETE REGIONS OF THE RAT BRAIN STEM

—Choline acetyltransferase (ChAc) content of 50 separate rat brain stem nuclei and cerebellum removed by microdissection was determined using a sensitive radiometric assay. The distribution of ChAc activity is uneven, with extremely high levels in the cranial motor nuclei and the nucleus salivatorius. Low ChAc concentrations were observed in the cranial sensory nuclei, the nuclei of the reticular formation, the raphe nuclei and the nuclei of the acoustic system. The lowest ChAc levels were measured in the cerebellum. Comparison of the distribution of ChAc with histochemical localization of acetylcholinesterase revealed generally good agreement, and notable exceptions are discussed.     

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.     

CONTROL OF ACETYLCHOLINE SYNTHESIS—THE INHIBITION OF CHOLINE ACETYLTRANSFERASE BY ACETYLCHOLINE

Abstract— The inhibition of choline acetyltransferase by acetylcholine in vitro occurs at a concentration of 10 m m and increases progressively to 45 per cent at a concentration of 100 m m . The inhibition is competitive for choline and noncompetitive for acetyl-CoA. It is suggested that the synthesis of acetylcholine may be controlled by its accumulation in synaptic vesicles.