Isolation and various properties of alpha-aminocaprolactam hydrolase from Klebsiella aerogenes]

L-alpha-aminocaprolactam hydrolase possessing the L lysine amidase activity was isolated from Klebsiella aerogenes and purified. The procedure of enzymes purification included cell destruction on USDN-I, fractionation by ammonium sulfate, gel chromatography on G-200. The preparation of the purified enzyme possessed specific activity of 50 mumol of lysin per 1 mg of protein per hour. Km was 2.6 mM in case of phosphate buffer (ph 7.2) for I-alpha-aminocaprolactam. Besides L-alpha-aminocaprolactam the enzyme hydrolyses lysine amide, leucine amide tryptophanamide. Magnesium ions are necessary for manifestation of catalytic activity of the enzyme.     

Purification of acyl hydrolase enzymes from the leaves of Phaseolus multiflorus

Acyl hydrolase activities have been purified from the leaves of Phaseolus multiflorus. The purification procedure involved heat treatment, DEAE-cellulose chromatography, Sephadex G-100 filtration and hexyl agarose chromatography. The elution pattern from hexyl agarose columns together with substrate competition experiments indicated the presence of two hydrolase enzymes. The first could hydrolyse oleoylglycerol and phosphatidylcholine while the second would deacylate glycosylglycerides and oleoylglycerol. Overall purification of both enzymes was ca 70-fold and the MW of the glycosylglyceride-hydrolysing enzyme was in the range 70–78000.     

Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex.

Sterol ester hydrolase (cholesterol esterase, E.C. 3.1.1.13) of bovine adrenal cortex has been extensively purified by ammonium sulfate fractionation, acid precipitation, hydroxylapatite chromatography, and Sephadex G-200 chromatography. During the purification sequence, the hydrolase activity was purified free of endogenous protein kinase. With this purified preparation, activation by cyclic AMP and ATP-Mg2+ did not occur unless exogenous protein kinase was included in the activating system. Using [gamma-32P]ATP, the transfer of the terminal phosphate to the enzyme protein was demonstrated by three separate experimental approaches. With pooled fractions from Sephadex G-200 chromatography, significant binding of 32P by the enzyme protein was observed only in the presence of exogenous protein kinase. Time course studies disclosed a close concurrence between the extent of activation of the purified enzyme by cyclic AMP-dependent protein kinase and the level of 32P transfer from [gamma-32P]ATP to the enzyme protein. Finally, assays carried out during Sephadex G-200 chromatography showed a correspondence in the peaks for activated sterol ester hydrolase and for 32P binding by protein. The data confirm that activation of adrenal sterol ester hydrolase by cyclic AMP and ATP-Mg2+ involves protein kinase-catalyzed phosphorylation of the enzyme protein.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Purification and Some Properties of Maleylpyruvate Hydrolase and Fumarylpyruvate Hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn²⁺ ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.     

Purification of rat pancreatic lipase

A procedure for the isolation of lipase (glycerolester hydrolase, EC 3.1.1.3) from rat pancreas is described. The purification scheme includes homogenization of the pancreas, centrifugation at 3,000 rpm, centrifugation at 40,000 rpm, DEAE-cellulose chromatography, precipitation of amylase as the amylase-glycogen complex, gel filtration of the amylase-free proteins on Sephadex G-100, and chromatography on carboxymethyl-Sephadex C-50. The enzyme showed only one band on polyacrylamide gel electrophoresis and had a specific activity of 5330 +/- 80 units/mg of protein.     

ACETYLCHOLINESTERASE IN MOUSE BRAIN, ERYTHROCYTES AND MUSCLE

Abstract— The properties of the enzyme acetylcholinesterase (EC 3.1.1.7) from mouse brain, erythrocytes and muscle were investigated. The enzymes were examined by gel filtration in Sephadex G-200, polyac-rylamide gel electrophoresis, immunological reactions, active-site labelling with tritiated di-isopropyl-phosphorofluoridate and also their kinetic properties were compared. All three enzymes appeared to have a single active small mol. wt. component of 80,000 to 82,000 which produced higher mol. wt. forms by aggregation. The partial purification of the enzyme from brain was achieved by affinity chromatography and this product was used to prepare antibodies. The purified immunoglobulin was shown to react with all three enzymes.     

Problems associated with the assay of arylsulphatases A and B of rat tissues

A method for the separation and purification of rat liver arylsulphatases A and B by gel filtration on Sephadex G-200 is described. The properties of the A enzyme and its molecular weight are similar to those of the corresponding ox liver enzyme. The B enzymes were found to be dissimilar. The method already developed for the assay of the corresponding enzymes from human tissues was shown to be unsuitable for the assay of the enzymes of rat tissues. A method of assay was developed which permits an approximate determination of the individual rat liver enzymes in a mixture of the two, but precise determination requires prior separation of the enzymes by gel-filtration chromatography.     

Characterization of a new extracellular hydrolase from Thermobifida fusca degrading aliphatic-aromatic copolyesters

The paper describes the purification, biochemical characterization, sequence determination, and classification of a novel thermophilic hydrolase from Thermobifida fusca (TfH) which is highly active in hydrolyzing aliphatic-aromatic copolyesters. The secretion of the extracellular enzyme is induced by the presence of aliphatic-aromatic copolyesters but also by adding several other esters to the medium. The hydrophobic enzyme could be purified applying a combination of (NH(4))SO(4)-precipitation, cation-exchange chromatography, and hydrophobic interaction chromatography. The 28 kDa enzyme exhibits a temperature maximum of activity between 65 and 70 degrees C and a pH maximum between pH 6 and 7 depending on the ion strength of the solution. According to the amino sequence determination, the enzyme consists of 261 amino acids and was classified as a serine hydrolase showing high sequence similarity to a triacylglycerol lipase from Streptomyces albus G and triacylglycerol-aclyhydrolase from Streptomyces sp. M11. The comparison with other lipases and esterases revealed the TfH exhibits a catalytic behavior between a lipase and an esterase. Such enzymes often are named as cutinases. However, the results obtained here show, that classifying enzymes as cutinases seems to be generally questionable.     

一种纯化细菌有机磷水解酶的简便方法

Cibacron blue T_3GA与溴化氰活化的Sepharose 4B偶联后,产生一种能有效地分离有机磷水解酶的吸附剂。用0.15mol/L MgCl_2溶液从黄杆菌P3—2细胞抽提出的粗酶液通过柱层析分离,即可得到纯化8倍、酶活性回收率为269.4％的纯酶制品。该酶制品用凝胶电泳测是均一的。     

Purification of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli strain 080

Purification studies of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 degrees C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54,000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera.     

Purification of β-acetylglucosaminase and β-galactosidase from ram testis

1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.     

Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase.     

Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase.     

Purification and properties of cholesterol ester hydrolase from human aortic intima and media.

1. Cholesterol ester hydrolase of human aortic intima and media was isolated and purified about 650-fold with 10-15% recovery of the original activity by sequential precipitation with 35% acetone, gel filtration on Sephadex G-75 and DEAE-cellulose column chromatography. 2. Two pH optima of 4.5-5.0 and 7.0-7.5 were consistently observed for the partially purified cholesterol ester hydrolase of human aortic intima and media. 3. In the system used in the present study, the increasing concentration of emulsifiers, sodium taurocholate and phosphatidylcholine, inhibited the activity of the neutral enzymes but not on the acid enzymes. On the contrary, reaction products, cholesterol and oleic acid, were much more inhibitory on the acid enzymes than on the neutral ones. 4. Results of studies on the effect of presentation of substrate on the enzyme activity and on the difference between acid and neutral enzymes are also discussed.     

Characterization of Bovine Atrial Angiotensin-Converting Enzyme

Bovine atrial angiotensin-converting enzyme (ACE) was purified to electrophoretic homogeneity. The purification procedure included ion-exchange chromatography on DEAE-Toyopearl 650M, affinity chromatography on lisinopril-agarose and gel filtration on Sephadex G-100. The bovine atrial ACE exhibited similar sensitivities to inhibition by lisinopril and captopril as lung ACE (the K _i values for the atrial and lung enzymes differed insignificantly). However, the kinetic parameters of hydrolysis of some synthetic tripeptide substrates (FA-Phe-Gly-Gly, FA-Phe-Phe-Arg, Cbz-Phe-His-Leu, Hip-His-Leu) catalyzed by bovine atrial and lung ACE varied to a greater extent. The enzymes were also characterized by some differences in activation by chloride, nitrate, and sulfate anions. These data support the hypothesis of tissue specificity of ACEs.     

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.     

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.     

Myrosinase in Brassicaceae: I. LOCALIZATION OF MYROSINASE IN CELL FRACTIONS OF ROOTS OF Sinapis alba L.

Myrosinases (thioglucoside glucohydrolases E.C. 3.2.3.1 [EC] .), whichcatalyse the hydrolysis of glucosinolates present in Brassicaceae,were isolated from Sinapis alba L. seeds. The crude enzyme extractwas purified using gel and ion-exchange chromatography, isoelectricfocusing, and polyacrylamide gel electrophoresis. The separationof two myrosinase isoenzymes was obtained after gel chromatographyon Sephadex G-100. Further purification of the main myrosinasecomponents was achieved when the combined isoenzymes were separatedon the anion-exchanger DEAE-Sephadex A-50 followed by polyacrylamidegel electrophoresis. A similar purification was obtained when the crude extract wasgroup-fractionated on Sephadex G-50 followed by DEAE-cellulosechromatography on Whatman DE-52 and gel chromatography on SephadexG-200. The enzyme from the last step was further separated byisolectric focusing into two isoenzymes with isoelectric points4.9 and 6.2. In order to clarify where the myrosinase was localized in theroot tip cells, cell fractionation studies were performed usingaldehydes as pre-fixatives to stabilize the enzymes and thecell organelles. Biochemical tests of crude and purified samplesof the isolated myrosinases showed that when glutaraldehydeor formaldehyde were used as pre-fixatives at a final concentrationof 1% (w/v), they did not inhibit the enzyme activity. Relativelyhomogeneous cell organelle fractions were obtained using ultracentrifugationand stepwise sucrose gradients. The myrosinase activity expressedon the basis of the protein content was found to be highestin the dictyosome and smooth endoplasmic reticulum fractions     

Simple and unique purification by size-exclusion chromatography for an oligomeric enzyme,rat liver cytosolic acetyl-coenzyme A hydrolase

An overview of the purification of an oligomeric enzyme, an extramitochondrial acetyl-coenzyme A hydrolase from rat liver, is presented. The enzyme has been purified to homogeneity using two successive size-exclusion chromatography runs, first for the monomeric and second for the oligomeric form of the enzyme. The sequential gel-filtration steps efficiently removed the contaminants of any molecular size, first of different size from that of the monomeric form of the enzyme (K(av)=0.47 on Superdex 200) and second of different size from that of the oligomeric form (K(av)=0.33), allowing us to purify the enzyme in high purity. This strategy provides an excellent model for purifying many other oligomeric proteins including key enzymes or allosteric enzymes regulating metabolism.