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.     

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.     

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.     

CHARACTERIZATION OF HIGH AFFINITY CHOLINE UPTAKE BY TORPEDO CALIFORNICA T-SACS

Abstract— The high affinity choline uptake system present in T-sacs prepared from Torpedo californica electric organ was shown to be insensitive to external Ca²⁺ and to be absolutely dependent on external NaCl, with optimal uptake at approx 200 mM-NaCl. Both Na⁺ and Cl⁻ separately stimulate uptake. Uptake also exhibited an optimum at approx 10mM-external K⁺. Uptake was completely inhibited at 4°C. Approximately 50% of newly accumulated [³H]choline was released by depolarization of T-sacs regardless of the time or method of depolarization.     

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.     

MOLECULAR PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM DIFFERENT SPECIES INVESTIGATED BY ISOELECTRIC FOCUSING AND ION EXCHANGE ADSORPTION

—The isoelectric point, surface charge and K_m for choline of choline acetyltransferase from different species were determined. Choline acetyltransferase from mouse and monkey brain was resolved into three molecular forms with isoelectric points at 7·1, 7·5, 8·4 and 7·0, 7·35, 8·35 respectively, whereas choline acetyltransferase from the electric organ of Torpedo and from rabbit brain showed a molecular form with isoelectric point 6·6 and 6·9, respectively. With the exception of rabbit brain enzyme, there was a good correlation between the isoelectric points and surface charges of the different choline acetyltransferases. The K_m's for choline were 0·66, 0·88, 0·92 and 3·5 mM for monkey, mouse, rabbit and Torpedo choline acetyltransferase respectively. The separated molecular forms of mouse and monkey enzymes did not show any significant difference in their affinity for choline.     

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 CATION SENSITIVITY OF THE ACETYLCHOLINE RECEPTOR FROM TORPEDO CALIFORNICA

—The effects of various cations and cholinergic ligands on the rate of α-bungarotoxin-acetylcholine receptor complex formation has been studied by means of a DEAE-cellulose disk assay technique. The reaction rate is reduced to one half the initial rate in the presence of 2·5 · 10^?6m acetyleholine. a value close to the observed K_D of ACh measured by equilibrium dialysis. Inhibition constants of about 5 mM were obtained for most monovalent cations whereas divalent cations gave inhibition constants of 0·05-0·2 mm . The rate and extent of toxin-receptor complex formation was also investigated as a function of hydrogen ion concentration; the rate of formation reaches a maximum at pH 7·5 and a group with a pK about 6 inhibits toxin binding to the receptor when protonated. These data can be correlated with the observed effects of inorganic cations on the binding of cholinergic ligands in vivo at the neuromuscular junction. Given the affinities of the individual cations, it is possible to predict how the apparent affinity of a cation-sensitive cholinergic ligand will change with variations of buffer composition.     

EFFECT OF SALTS ON THE PHYSICAL AND KINETIC PROPERTIES OF HUMAN PLACENTAL CHOLINE ACETYLTRANSFERASE

Abstract— The effects of salt on the properties of human placental choline acetyltransferase have been examined. Increases in enzyme activity, thermal denaturation and susceptibility to proteolysis can be related to increases in ionic strength, rather than to specific salt effects. Increased ionic strength increases the maximal velocity (K_m) of the reaction, with no change in the kinetic parameter V_max/K_m (choline). The pH-K_m profile, measured over the range of 6.5–8.0, indicates the requirement of a dissociated acidic residue whose pKa is below 7.5 at high ionic strength, and a protonated residue whose pKa is above 7.5 at low ionic strength. It is proposed that the conformation of the enzyme is different at high ionic strength and at low ionic strength, and that these different conformational states of the enzyme result in different rate-determining steps of the reaction.     

MOLECULAR CHARACTERIZATION OF CHOLINE ACETYLTRANSFERASE FROM BOVINE BRAIN CAUDATE NUCLEUS AND SOME IMMUNOLOGICAL PROPERTIES OF THE HIGHLY PURIFIED ENZYME

Choline acetyltransferase from bovine brain caudate nucleus has been purified to a specific activity of 25–30 μmol ACh formed per min and mg protein. Disc electrophoresis at pH 9.5 of the purified enzyme showed two protein bands localized close to each other. We were not able to show if ChAT was linked to one or both bands. In SDS disc electrophoresis the enzyme preparation showed one major and one minor protein band with molecular weights of 69,000 and 34,000, respectively. Heterogeneity of the enzyme preparation was also demonstrated by immunodiffusion and immunoelectrophoresis. After ammonium sulphate precipitation no aggregation of the enzyme could be detected by gelfiltration on Ultrogel AC-34 whilst a high molecular weight fraction was occasionally observed by gelfiltration on Sephadex G-200. The enzyme was, however, separated into two molecular forms (A and B) on CM-Sephadex chromatography. Both molecular forms had the same S²_20w but differed in heat stability and affinity for acetyl-CoA. Both forms were inactivated by an antibody preparation raised against either a purified preparation of ChAT, or A and B separately. The highly purified enzyme preparation was inactivated more than 98% by immunoprecipitation. The antibody crossreacted with ChATs from several mammalian species, but only slightly with ChAT from pigeon. The results of binding studies with affinity columns, suggest that the enzyme contains a hydrophobic lobe and a dinucleotide fold, and that a free purine rather than a free ribosyl ring of acetyl-CoA is important for the binding of the substrate to the active site. The hydrophobic lobe may be the same as the dinucleotide fold.     

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.     

THE BINDING OF ACETYLCHOLINE TO SYNAPTIC VESICLES FROM TORPEDO CALIFORNICA ELECTROPLAX

Abstract— The in vitro uptake of exogenous acetylcholine by isolated presynaptic vesicles has been demonstrated in a new system. A preparation of vesicles from Torpedo californica electroplax was developed in which acetylcholinesterase and acetylcholine receptor activity were blocked. The vesicles bound acetylcholine with K_d 1.58 μM, the maximum amount bound being 26 pmol per g of original tissue, or 52 molecules per vesicle. Nicotinic drugs blocked binding, but muscarinic and noncholinergic drugs did not. The relative potency of nicotinic drugs differed greatly from their potency on Torpedo receptor. Sephadex chromatography showed that 26% of the binding was irreversible. The relationship of the binding to acetylcholine uptake and storage was discussed.     

PARTIAL PURIFICATION AND PROPERTIES OF CHOLINE KINASE (EC 2.7.1.32) FROM RABBIT BRAIN: MEASUREMENT OF ACETYLCHOLINE

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min^?1 mg^?l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS0₄, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm ) to phosphorylcholine [³²P] in the presence of ATP [y³²P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two K_m values for choline at pH 9.5; 32 μm and 0.31 mm . At pH 7.4, the higher K_m was not observed and enzyme activity was maximal with 0.1 mm choline. The K_m for ATP was 1.1 mm . Enzyme activity was inhibited by ATP (20 mm ), AMP, ADP, cytidine diphosphocholine (1 or 10 mm ), and activated by choline esters (1.0 mm ), NaCl or KCl(200 mm ).     

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.     

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.     

螺旋藻氢酶的纯化与生化特性

本研究用DE－52、SephadexG－75、SephadexG－100柱层析从螺旋藻分离纯化得到比活性提高200倍的氢酶,回收率为14％。凝胶柱层析和SDS－PAGE显示一条带,其分子量为56kd。氨基酸分析结果表明酸性氨基酸比例较大,等电聚焦测定结果证明其等电点为pH4．2。吸收光谱结果显示氢酶是铁硫蛋白。甲基紫晶（MV）是氢酶催化放氢的最佳电子供体,其Km（MV）为0．31mmol／L,最适pH值为7．5－8．0。     

黑曲霉纤维素酶系中β-葡聚糖纤维二糖水解酶的提纯和性质

通过硫酸铵分段,Sephadex G-100凝胶过滤及DEAE-SephadexA-25(A-50)离子交换柱层析等提纯步骤,从黑曲霉(Aspergillus niger)培养液中分离到β-葡聚糖纤维二糖水解酶的两个组分(Ⅰ-3a,Ⅰ-3b),经凝胶电泳鉴定均为单一带,Ⅰ-3a和Ⅰ-3b的最适pH分别为4.0及4.5,最适温度50℃及45℃,在pH2.0—8.0之间稳定,保温1h时的半失活温度t 1/2分别为52℃及48℃。SDS-凝胶电泳法测得Ⅰ-3a和Ⅰ-3b的分子量分别为57000及55000;聚丙稀酰胺凝胶等电聚焦法测得二者的等电点分别为3.2和3.0。在所测定的化学试剂中,Ag~+、Hg~(2+)和Cu~(2+)对该酶均有较强的抑制作用。     

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.     

黄杆菌(Flavobacterium sp.)几丁质酶的纯化和性质

黄杆菌(Flavobacteium sp.)在几丁质的诱导下产生几丁质酶.通过(NH_4)_2SO_4沉淀、DEAE纤维素柱层析、Sephacryl 300柱层析及Sephadex G-75柱层析,从Flavobacterium sp.培养上清液中分离纯化了几丁质酶.SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯度分析表明,纯化后的几丁质酶达到了均一的程度.用SDS-PAGE测得该酶的分子量约45D00道尔顿.该酶水解几丁质的最适pH为 7.0,最适温度为50℃,-20C贮存两年以上仍有活性.水解几丁质的Km值为5.0mg/ml.金属离子对几丁质酶活性影响较大,Ca^(2+) 、Co^(2+)’和Cu^(2+)对酶有激活作用.而NH_4^-、Ba^(2+)、Mg^(2+)、Mn^(2+)对酶有抑制作用.几丁质酶水解几丁质的产物是几丁质二糖.     

夏威环毛蚓纤溶酶的分离纯化及部分性质研究

以夏威环毛蚓（Ｐｈｅｒｅｔｉｍａ ｈａｗａｙａｎａ）为材料,采用磷酸盐缓冲液抽提、（ＮＨ４）２ＳＯ４分段盐析,离子交换树脂 Ｄ２９０、 Ｓｅｐｈａｄｅｘ Ｇ－１００和 ＤＥＡＥ－ｓｅｐｈａｄｅｘ Ａ－５０三种连续柱层析方法得到一种在 ＰＡＧＥ上显示单一区带的纤溶酶组份。采用凝胶柱层析和 ＳＤＳ－ＰＡＧＥ测其分子量为 １２ ０００和 １２３００,由一条肽链组成。该酶具有强烈的纤溶活力和水解ＢＡＥＥ的活力,能直接作用纤维蛋白和间接激活纤溶酶原。其最适反应温度为４５℃,最适反应ｐＨ为８．０。该酶水解ＢＡＥＥ的活力可被Ｎａ＋、Ｋ＋、Ｍｇ２＋、Ｈｇ２＋、金属离子和ＥＤＴＡ、巯基乙醇抑制,Ｃａ＋则有激活作用。该酶中性糖含量为５％,氨基酸组成中Ａｒｇ、Ｌｅｎ含量较多．