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.     

A FLUOROMETRIC ASSAY FOR CHOLINE ACETYLTRANSFERASE and ITS USE IN THE PURIFICATION OF THE ENZYME FROM HUMAN PLACENTA

Abstract— A fluorometric assay for choline acetyltransferase has been developed. This assay is based on coupling the choline acetyltransferase dependent formation of acetyl-CoA from acetylcholine and coenzyme A, to the reactions catalyzed by the enzymes citrate synthase and malic dehydrogenase. Although this assay is not as sensitive as previously described radiometric assays, it can be conveniently used during enzyme purification.
Employing this assay method, choline acetyltransferase has been purified from human placenta to a specific activity of 92.7 μmol acetylcholine formed/min/mg protein.     

Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase

Abstract: A cDNA for Drosophila choline acetyltransferase (EC 2.3.1.6; ChAT) was fused with a polyhistidine sequence and expressed in Escherichia coli. The recombinant enzyme was purified to a specific activity of 500 μmol/min/mg of protein using metal affinity chromatography and ion exchange chromatography. Kinetic properties of the recombinant enzyme did not differ significantly from those previously determined. Circular dichroism (CD) spectra revealed that the secondary structure of the enzyme is largely μ-helical. Intrinsic fluorescence spectra of the enzyme indicate that its tryptophan residues are buried. Neither CD nor fluorescence spectra changed significantly in the presence of substrates. The cysteine content of the recombinant Drosophila ChAT was determined to be 16 in the absence and 22 in the presence of 6 M guanidine hydrochloride. Finally, crystallization of recombinant Drosophila ChAT was achieved.     

Immunological properties of rat brain choline acetyltransferase.

Abstract— Four antisera active against choline acetyltransferase (ChAc) were obtained by injecting 22 rabbits with rat brain ChAc. The ChAc preparations used for immunization (specific activity from 015 to 2 μmol/min/mg of protein) were not pure and the antisera produced were not monospecific. The antisera inhibited and precipitated ChAc, but the precipitated enzyme-antibody complexes still retain ChAc activity. One millilitre of the most active serum precipitates 0–5 μmol/min of rat brain ChAc at the equivalence point. Its titre expressed in mg/ml of immunoglobulins precipitated with the antigen and the equivalence point was calculated at about 0.08 mg/ml of serum. This relatively low titre explains the lack of any visible ChAc immunoprecipitate in an immunodiffusion test. Cross-reactivity studies revealed that ChAc has undergone few changes during evolution, since antisera produced against rat brain ChAc still precipitate ChAc from fish (Torpedo).     

CHOLINE ACETYLTRANSFERASE–EVIDENCE FOR ACETYL TRANSFER BY A HISTIDINE RESIDUE

Abstract— Choline acetyltransferase from bovine brain has been extensively purified to a specific activity of 2.5 μmol ACh/min mg protein. Attempts to isolate an acetyl enzyme intermediate after incubation of the enzyme with [1-¹⁴C]acetyl-CoA were unsuccessful. Such an intermediate could only be isolated using a 30-fold less purified enzyme preparation. The protein, binding ¹⁴C in this preparation, did not correspond to choline acetyltransferase as shown by disc-electrophoresis. The highly purified enzyme could, however, be labelled when choline acetyltransferase was immobilized on a mercuribenzoate sepharose gel and incubated with [1-¹⁴C]acetyl-CoA. Subsequently, the immobilized labelled enzyme or the labelled enzyme which had been released by cysteine from the gel. formed ACh after incubation with choline. The labelling and the following formation of [¹⁴C]ACh was pH dependent.
Masking htstidine residues of the enzyme with diethylpyrocarbonate almost abolished the labelling of the immobilized enzyme and completely abolished the formation of [¹⁴C]ACh. Enzyme inhibited with 5.5'-dithiobis(2-nitrobenzoate) was partially reactivated when the thionitrobenzoatederivative was cleaved by KCN treatment to a thiocyanatederivalive. A reaction mechanism for ChAT is proposed based on the present data.     

Evidence for the extreme overestimation of choline acetyltransferase in human sperm, human seminal plasma and rat heart: a case of mistaking carnitine acetyltransferase for choline acetyltransferase

Detection of choline acetyltransferase (ChAc) in a number of non-neuronal tissues has been extremely overestimated. There are two major types of errors encountered. Type 1 error occurs when endogenous substrates (e.g. L-carnitine) are acetylated by acetyltransferase enzymes (e.g. carnitine acetyltransferase ( CarAc ) ) yielding an acetylated product mistaken for acetylcholine (AcCh). In the past, human sperm and human seminal plasma putative ChAc activity has been extremely overestimated due to Type 1 error. This study demonstrates (1) an endogenous acetyltransferase and substrate activity in human sperm and human seminal plasma forming an acetylated product that is not AcCh but probably acetylcarnitine ( AcCar ); (2) that the addition of 5 mM choline substrate does not significantly increase acetyltransferase activity; (3) that boiled seminal plasma contains an endogenous acetyltransferase substrate which is not choline, but probably L-carnitine. Type 2 error occurs when endogenous carnitine acetyltransferase synthesizes true AcCh, resulting in mistaken evidence for ChAc. This is demonstrated by the fact that the choline substrate Km-value for the neuronal or true ChAc from mouse brain is 0.73 +/- 0.06 mM while the Km-value of choline substrate for purified CarAc from pigeon breast muscle is 108 +/- 4 mM. Type 2 error has occurred for the estimation of putative ChAc in rat heart. The rat heart ChAc was measured in previous studies utilizing a concentration of 30 mM choline substrate. While saturation of neuronal ChAc is observed at 2-5 mM choline, saturation of the rat heart CarAc enzyme is not reached until over 800 mM. Purified CarAc significantly synthesizes AcCh at 30 mM choline. Thus, putative ChAc has been greatly overestimated in the scientific literature for mammalian sperm, human seminal plasma and rat heart.     

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.     

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-CoA, ACETYL-DEPHOSPHO-CoA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K_m values of 25.7 μ m for acetyl-CoA, 54.8 μ m for acetyl-dephospho-CoA and 382 μ m for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.
Inhibitor constants ( K_i ) for CoA, dephospho-CoA and pantetheine phosphate were also measured and when considered with the K_m values obtained for the acetyl derivatives it is concluded that acetyl-dephospho-CoA could be a successful acetyl donor in the synthesis of ACh.
Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.     

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 of chicken brain choline acetyltransferase

Chicken brain choline acetyltransferase was purified to homogeneity using ammonium sulfate fractionation, followed by chromatography on DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150, immunoabsorption and Sepharose-CoA columns. A purification of 3500-fold was achieved and the final preparation had a specific activity of 2:32 μmol acetylcholine formed per minute per milligram protein. The purified chicken choline acetyltransferase migrated as a single band on polyacrylamide gel electrophoresis in the presence and absence of sodium deodecyl sulfate. The native enzyme, with a molecular weight of 67,000 daltons, consists of two subunits of identical molecular weight. Chicken choline acetyltransferase has a sharp pH optimum of 7.4. It is activated by sodium chloride and potassium chloride but inhibited by cupric ion and N-ethylmaleimide.     

Inhibitory actions of muscarinic cholinergic receptor agonists on serotonin N-acetyltransferase in bovine pineal explants in culture

We have previously identified muscarinic cholinergic receptors in the bovine pineal gland with a K_D value of 0.423±0.01 nM and a B_max value of 69.75±20.91 fmol/mg protein. Similarly, we have shown that the bovine pineal gland possesses a specific choline acetyltransferase with an activity of 0.034±0.004 nmol/mg protein/min. In order to delineate the function of these cholinergic receptor sites, we have studied the effects of muscarinic cholinergic receptor agonists on the activity of serotonin N-acetyltransferase, the melatonin synthesizing enzyme. Cholinergic receptor agonists such as methacholine (10 M), carbachol (10 M), and oxotremorine (10 M) inhibited the activity of serotonin N-acetyltransferase in the bovine pineal explants in culture, from a control value of 5.02±0.45 to 1.25±0.25, 1.30±0.15, and 1.22±0.20 pmol/mg protein/min, respectively. These inhibitory effects were blocked by muscarinic cholinergic receptor antagonists such as atropine (20 M) or QNB (20 M). The presence of high affinity muscarinic cholinergic binding sites, of a specific choline acetyltransferase, along with an inhibitory action of cholinomimetic agents on the activity of serotonin N-acetyltransferase, are interpreted to suggest that muscarinic cholinergic fibers may modulate the synthesis and actions of pineal melatonin.     

Biophysical properties of rat brain choline acetyltransferase.

Abstract— The molecular weight of choline acetyltransferase (ChAc) was determined by using a variety of methods. A molecular weight of 68,000 daltons was found. ChAc is a globular protein with an apparent radius of 3.39 nm (value of the Stokes radius). The existence of a dimeric form and higher aggregates is discussed     

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholin-ergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipi-tate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with ³²P_i incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μ M veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.     

Isolation,purification, and characterization of haloalkaline xylanase from a marine <Emphasis Type="Italic">Bacillus pumilus</Emphasis> strain,GESF-1

A haloalkalitolerant xylanase-producing Bacillus pumilus strain, GESF1 was isolated from an experimental salt farm of CSMCRI. Birch wood xylan and xylose induced maximum xylanase production with considerable activity seen in wheat straw and no activity at all with caboxymethyl cellulose (CMC). A three step purification yielded 21.21-fold purification with a specific activity of 112.42 U/mg protein (unit expressed as μmole of xylose released per min). Xylanase produced showed an optimum activity at pH 8.0, with approximately 50 and 30% relative activity at a pH 6.0 and 10.0, respectively. The temperature optimum was 40°C and kinetic properties such as K_m and V_max were 5.3 mg/mL and 0.42 μmol/min/mL (6593.4 μmol/min/mg protein). Xylanase activity (160∼ 120%) was considerably enhanced in 2.5 to 7.5% NaCl with 87 and 73% retention of activity in 10 and 15% of NaCl. Enzyme activity was enhanced by Ca²⁺, Mn²⁺, Mg²⁺, and Na⁺ but strongly inhibited by heavy metals such as Hg²⁺, Fe³⁺, Cu²⁺, Cd²⁺, and Zn²⁺. Organic reagents such as β-Mercaptoethanol enhanced xylanase activity whereas EDTA strongly inhibited its activity. Xylanase, purified from the Bacillus pumilus strain, GESF1 could have potential biotechnological applications.     

DISTRIBUTION OF ACETYLCHOLINE, CHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN REGIONS AND SINGLE IDENTIFIED AXONS OF THE LOBSTER NERVOUS SYSTEM

Abstract— Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine-hydrolysing enzyme is largely membrane-bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X-100-treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500-fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23-fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20–2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline ( ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1·9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500-fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.     

REGIONAL AND SUBCELLULAR DISTRIBUTION AND KINETIC PROPERTIES OF RAT BRAIN CHOLINE ACETYLTRANSFERASE–SOME FUNCTIONAL CONSIDERATIONS

The kinetic properties of soluble and membrane-bound choline acetyltransferase (ChAc) were determined as a function of homogenization media and solubilization procedure in various regions of rat brain. Treatment of homogenate and/or subcellular fractions with KCl, Triton X-100, or ether dramatically altered the apparent V_max and the degree of solubilization of the enzyme, but no fraction exhibited K_m values substantially different from 12 μM for acetyl-CoA and 200 μM for choline. On the other hand, increasing the ionic strength of the assay medium for a given fraction from 0-02 M to 0-5 M increased both V_max and K_m values for both substrates. The absolute levels and subcellular distribution of ChAc were determined in 11 brain regions to localize cholinergic cell bodies and nerve endings. Levels of ChAc varied from 139 m-units/g tissue in caudate-putamen to 5-7 m-units/g tissue in cerebellum. The fraction of ChAc activity associated with synaptosomes varied from near 75 per cent in caudate-putamen, hippocampus and cortical regions to near 20 per cent in septum, locus coeruleus area and substantia nigra area. The apparent parallel distribution of cholinergic and catecholaminergic nerve endings is discussed in terms of a hypothetical model for the pathophysiology and treatment of Parkinson's syndrome.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Purification and characterization of soybean nodule nitrite reductase

A nodule cytosol nitrite reductase was isolated from soybean [ Glyine max (L.) Mer. cv. Tracy] grown in the presence of nitrate. Enzyme activity increased when increased amounts of nitrate were supplied to the plant. A purification procedure involving ammonium sulfate precipitation, gel filtration, DEAE Sephadex and Blue Sepharose chromatography resulted in an activity capable of forming 6.7 μmol ammonia (mg protein)⁻¹ min⁻¹. This represented a 235-fold increase in specific activity. The molecular weight, estimated by gel filtration, was 55 000. The pH optimum for activity was 7.1. Ammonia formed stoichiometrically as nitrice was consumed. From Lineweaver-Burk plots, K_m values of 0.5m M for nitrite and 0.2m M for methyl viologen were calculated. Spectral data suggest the association of a heme chromophore with the enzyme.     

THE SYNTHESIS OF ACETYLCHOLINE BY THE OLIVOCOCHLEAR BUNDLE

Abstract— Choline acetyltransferase (ChAc; EC 2.3.1.6) was assayed in the membranous cochlea and in the eighth cranial nerve (both the vestibular and cochlear components) from the point where it leaves the brain stem to the internal auditory meatus of the cat. To determine the contribution of the efferent innervation of the cochlea to this enzymatic activity both eighth nerves and both membranous cochleae were assayed at 17–29 days following section of the right, crossed and uncrossed olivo-cochlear bundles (OCB) in the cat. The lesion was produced along the right sulcus limitans on the floor of the fourth ventricle. The left eighth nerves and cochleae served as controls in the ChAc assay. There was a significant decrease in ChAc activity in the right cochlea and eighth nerve after OCB section and degeneration. The mean activity of ChAc in the right cochleae of the 6 operated cats was 15 ± 7 μg of ACh formed. h⁻¹ (g wet wt. of tissue) ⁻¹ in comparison to the rate of all the intact cochleae of 156 ± 38 μg of ACh formed. h⁻¹. (g of tissue)⁻¹, a statistically significant difference ( P < 0005). The mean activity of ChAc in the right eighth nerves of the cats with OCB lesions was 30 ± 8 n-g of ACh formed. h⁻¹. (g of tissue)⁻¹in comparison to 91 ± 19 fig of ACh formed . h⁻¹. (g of tissue)⁻¹ found for intact eighth nerves. This difference was also significant ( P < 0005). Thus, section and degeneration of the crossed and uncrossed OCB reduce the activity of ChAc in the eighth nerve and membranous cochlea. This finding provides support for the hypothesis suggesting the cholinergic nature of olivo-cochlear transmission.     

Regulation of Phospholipid Metabolism in Differentiating Cells from Rat Brain Cerebral Hemispheres in Culture: Ontogenesis of Carrier-specific Transport of Choline and N-Methyl-substituted Choline Analogs

Abstract: Dimethylaminoethanol was studied both as a substrate and as an inhibitor of choline uptake in long-term cultures of foetal rat cerebral hemispheres. A saturable component with an apparent K_m of 28 μM and V_max of 11 pmol/min/μg DNA for dimethylaminoethanol, was observed. Like choline, dimethylaminoethanol was also taken up by a second, low-affinity component, the apparent V_max of which was about 102 pmol/min/μg DNA. Dimethylaminoethanol inhibited the high-affinity but not the low-affinity choline uptake in a competitive manner with an apparent inhibition constant of 6.0 μM. Monomethylaminoethanol (K₁# 60 μM) competitively inhibited high-affinity choline transport. At low concentrations hemicholinium-3, but not ethanolamine, effectively inhibited high-affinity uptake of choline and to a lesser degree the uptake of the dimethylaminoethanol. While the high-affinity uptake of both substrates was inhibited by high concentrations of hemicholinium-3 or ethanolamine, the low-affinity system was not affected by hemicholinium-3. From the kinetics of uptake and inhibition patterns of choline and its related analogs, the methyl group seems to play a major role in determining the affinity rate constants for these substrates. The maximum rate of choline uptake via the high-affinity component increases about sixfold during a period of 2 weeks. In the absence of serum the maximum velocity of the high-affinity component is greatly reduced. These observations suggest that the high-affinity choline uptake component is an integral property and a useful marker, of the developing cerebral cells.