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 AND PROPERTIES OF BRAIN ALKALINE PHOSPHATASE

Abstract— Alkaline phosphatase from sheep brain has been purified to homogeneity. The method includes butanol extraction, fractional ethanol precipitation, ion-exchange chromatography on DEAE-cellulose, and on DEAE-Sephadex followed by Sephadex G-200 filtration. By these steps, the enzyme is purified 22,920-fold with 15% recovery. The homogeneous enzyme is shown to be a sialoglycoprotein in nature. Neuraminidase treatment reduces the electrophoretic mobility of the enzyme. The enzyme shows pyridoxal phosphate phosphatase activity along with p -nitrophenylphosphate phosphatase activity. Both these compounds behave as mutual alternate competitive substrates. The general properties of the enzyme are described.     

THE PURIFICATION AND PROPERTIES OF PIG BRAIN CATECHOL-o-METHYLTRANSFERASE

Abstract— The previously reported affinity chromatography technique (G ulliver & T ipton , 1978 b ) has been adapted for the large-scale purification of pig brain catechol- o -methyltransfcrase. The enzyme prepared by this method is apparently homogeneous by polyacrylamide gel electrophoresis criteria and is stable for prolonged periods of storage at—5°C in 20% (v/v) glycerol. Kinetic measurements, molecular weight criteria and antiserum reaction suggest that the brain and liver forms of catechol- o -methyltransferase are very closely related, if not identical.     

黑曲霉木聚糖酶的纯化与性质

由凝胶电泳酶谱检测到黑曲霉１４９发酵液中存在两型木聚糖酶,依次为Ｘ－Ⅰ和Ｘ－Ⅱ．通过硫酸铵分级沉淀及ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ５０柱层析分别将Ｘ－Ⅰ、Ｘ－Ⅱ纯化到凝胶电泳均一。由ＳＤＳ一凝胶电泳和浓度梯度凝胶电泳测得Ｘ－Ⅰ和Ｘ－Ⅱ的分子量分别为３７ｋＤａ,７６ｋＤａ,２４ｋＤａ和２３ｋＤａ,Ｘ－Ⅰ具有亚基。二者的含糖量分别为２７６％和７．３％。Ｘ－Ⅰ和Ｘ－Ⅱ最适反应温度分别为５０℃和５５℃,ｐＨ为４６和５．２。在ｐＨ４．６～９．２和ｐＨ４．０～１０．０之间Ｘ－Ⅰ、Ｘ－Ⅱ活力稳定。５０℃保温２４ｈ,Ｘ－Ⅰ活力仍为１００％,而Ｘ－Ⅱ的活力已降为２．８％。ＨｇＣｌ２和ＡｇＮＯ３显著抑制Ｘ－Ⅰ、Ｘ－Ⅱ的活力。Ｘ－Ⅰ与Ｘ－Ⅱ水解不同来源的木聚糖,其产物有所不同。     

THE PURIFICATION AND PROPERTIES OF RAT BRAIN GLUTAMATE DEHYDROGENASE

—A method is described for the preparation of glutamate dehydrogenase in a highly purified form from rat brain. Only one protein band was detected when the enzyme was subjected to electrophoresis on SDS polyacrylamide gels. The rat brain enzyme was essentially identical to the rat liver enzyme with respect to electrophoresis on SDS polyacrylamide gels, immunochemical properties and most kinetic parameters. However, the brain enzyme was much less reactive with glutamate, was more sensitive to inhibition by haloperidol, and was considerably more stable than the liver enzyme.     

河蚌C-反应蛋白的分离纯化及其性质研究

用合成的CDPC-Sepharose-6B亲和层析吸附剂一步提纯河蚌C-反应蛋白(CRP)。纯化的CRP在SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦电泳鉴定时均显示为一条区带。其等电点(pI)为5.2,分子量约为310,000道尔顿,由六个亚基组成,亚基分子量约为51,000道尔顿。在280nm处的消光系数E_(1cm)~(1mg)/ml=1.2。河蚌CRP与C_多糖(CPS)发生的沉淀反应是钙依赖性的。本文对河蚌CRP的氨基酸组成及其它性质也作了研究。     

人脑醛糖还原酶的纯化和一些基本性质

使用DEAE纤维素柱层析、PBE-94层析聚焦、NADP~+-Sepharose 4B亲合层析及SephadexG-100凝胶过滤分离纯化了人脑醛糖还原酶。在DEAE层析中,用咪唑-HCI缓冲液替代了磷酸缓冲液,改善了分离效果。在聚丙烯酰胺及SDS聚丙烯酰胺凝胶电泳中,纯化的人脑醛糖还原酶均呈一条区带。它的pI为5.6,最适pH为6.5,分子量为36,000,底物特异性和氨基酸组成与其它哺乳动物的醛糖还原酶有相似性。开链式醛糖是醛糖还原酶的真正底物,它在开链式和半缩醛的平衡体系中占比例极小,因而推知醛糖还原酶对此底物有很高的K_(cat)和K_(cat)/K_m值,能有效地将它们还原成相应的醇。     

PURIFICATION AND PROPERTIES OF HUMAN BRAIN α-L-FUCOSIDASE

Human brain α-L-fucosidase has been extracted and the soluble portion has been purified 9388-fold with 25% yield by a two-step affinity chromatographic procedure utilizing agarose-epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all seven isoelectric forms of the enzyme were purified. Trace amounts of eight glycosidases, with hexosaminidase being the largest contaminant (1% by activity) were found in the purified α-L-fucosidase preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of molecular weight 51,000 ± 2500. The purified enzyme has a pH optimum of 4.7 with a suggested second optimum of 6.6. The apparent Michaelis constant and maximal velocity of the purified enzyme with respect to the p-nitrophenyl substrate are 0.44 mM and 10.7 μmol/min/mg protein, respectively. Ag²⁺ and Hg²⁺ completely inactivated the enzyme at concentrations of 0.1-0.3 mM. Antibodies made previously against purified human liver α-L-fucosidase cross-reacted with the purified brain α-L-fucosidase and gave a single precipitin line coincident with that from purified liver α-L-fucosidase. From all our studies it appears that at least the soluble portion of brain α-L-fucosidase is identical to human liver α-L-fucosidase.     

PURIFICATION AND PROPERTIES OF BRAIN N-ACETYL-β-HEXOSAMINIDASE A

—N-Acetyl-β-hexosaminidase A was purified to homogeneity from human and monkey brains by the conventional procedures followed by concanavalin A–Sepharose affinity chromatography. The optimal activity was observed at pH 4·5 for both enzyme preparations with both the aglycones N-acetylglucosamine and N-acetylgalactosamine derivatives. The K_m values for hexosaminidase A from monkey brain were 0·26 mm and 0·04 mm respectively for N-acetylglucosamine and N-acetylgalactosamine. K_m values obtained for glucosamine and galactosamine derivatives for the human brain hexosaminidase A were of the same order. The glycoprotein nature of the enzymes was established by the affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the enzyme molecule from monkey brain.     

PURIFICATION AND PROPERTIES OF A PROLYL ENDOPEPTIDASE FROM RABBIT BRAIN

Abstract— An enzyme with the specificity of a prolyl endopeptidase was purified about 880-fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl-peptide and peptidylprolyl-amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N -benzyloxycarbonylglycyl-L-prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N -benzyloxycarbonylglycyl-L-proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.     

PURIFICATION AND PROPERTIES OF MITOCHONDRIAL MONOAMINE OXIDASE IN BEEF BRAIN

Abstract— Monoamine oxidase was purified approximately 40-fold from beef brain mitochondria. The purification procedure involved extraction with a non-ionic detergent (Nonion NS-210) after heat treatment, ammonium sulphate fractionation, chromatographies on DEAE-cellulose and Sepharose 6B, and a continuous flow electrophoresis. A major component (enzyme 1) with a higher specific activity and a minor component (enzyme 2) with a lower specific activity were separated. Properties of both enzymes towards kynuramine including pH-optimum and K_m values were similar, but the enzyme 1 had the higher specific activity towards tyramine whereas that of enzyme 2 was towards normetane-phrine. Fluorescence spectra indicated that the enzyme 1 is a flavoprotein. Copper was not detected, and copper chelating agents did not inhibit the enzyme. p -Chloromercuribenzoate and JV-ethylmaleimide inhibited the enzyme, indicating the presence of the essential SH-groups.     

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．     

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.     

THE PURIFICATION AND PROPERTIES OF BOVINE PINEAL S-ADENOSYLMETHIONINE: N-ACETYLSEROTONIN O-METHYL TRANSFERASE

Abstract— Bovine pineal gland S-adenosylmethionine: N-acetylserotonin O-methyltransferase has been purified about 2800-fold using cell fractionation, ammonium sulphate treatment, Sephadex G-200 gel filtration and anion exchange chromatography. The enzyme has been found to be a polymer; the smallest unit observed had a mol. wt. of 21,800 and the other polymers' molecular weights were multiples of this figure. In the gland extract polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. were more abundant than the others; they showed also higher specific activity. One of the products of the reaction, S-adenosylhomocysteine was found to be a potent inhibitor, whereas the other product, melatonin, did not inhibit the bovine pineal gland enzyme, even at much higher concentrations. Homocysteic acid, cysteic acid, GSG and GSSG inhibited the enzyme. The required concentrations for this effect was 100 times higher than that of S-adenosylhomocysteine. The addition of GSH to the medium during purification led to complete loss of activity. Adenosine, homocysteine and other thio compounds had little or no effect. The enzyme was found to be activated by its substrates and also by certain anions. Among various organic acid salts, citric acid cycle intermediates were found to be good activators; their nonsubstituted analogues were not as effective. The activator effect of oxaloacetate and bicarbonate was the highest, and was brought about by relatively low concentrations of these anions (1–5 × 10^?3 M), hence their effect was considered specific. The degree of activation caused by oxaloacetate was decreased by increasing substrate concentrations and vice versa. The S-adenosylhomocysteine inhibition could not be reduced by increasing the substrate concentration; S-adenosylhomocysteine also inhibited the oxaloacetate-activated enzyme. These observations have been explained by the allosteric behaviour of the enzyme. The kinetic behaviour of various polymers was also investigated. The highest substrate and oxaloacetate activation and the highest S-adenosylhomocysteine inhibition was observed for polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. The K_m values for S-adenosylmethionine and ^N-acetylserotonin calculated for the oxaloacetate activated enzyme were also lower for these polymers than others.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM OX BRAIN STRIATE NUCLEI

Abstract—

• 1 Choline acetyltransferase was purified from ox brain striate nuclei by an extraction step at pH 5, cation-exchange chromatography, fractional precipitation with ammonium sulphate, and chromatography on Sephadex G-200. The enzyme was obtained free of deacylases and cholinesterases, at specific activities of 01-0-3 μmol acetylcholine formed per min per mg protein.
• 2 The enzyme was found to be a stable and relatively basic protein, with a molecular weight of 65,000.
• 3 In the catalysed reactions, , k₁, was about four times k₂, and the equilibrium constant was approximately 40. For the forward reaction, the Michaelis constant for each substrate was independent of the concentration of the other (choline = 0-75 mM; acetyl-CoA = 10 μM), whereas in the back reaction one substrate increased the affinity for the other (acetylcholine = 0-75-5 MM; CoA = 25-150 μM).
• 4 CoA inhibited acetylcholine synthesis by competing with acetyl-CoA (K₁, = 16 μM). Acetylcholine slightly inhibited the forward reaction (e.g. 45 per cent in 200 mM) without competing with choline or acetyl-CoA. These data indicate an ordered reaction mechanism; acetyl-CoA probably always binds before choline.