Purification and properties of carbonyl reductase from rabbit kidney

An enzyme catalyzing the metabolic reduction of acetohexamide, an oral antidiabetic drug, has been purified from the cytosolic fraction of rabbit kidney to apparent homogeneity by various chromatographic techniques. The purified enzyme consists of a single polypeptide chain with a molecular weight of 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme requires NADPH as a cofactor and has an optimal pH of 6.0. A variety of xenobiotic carbonyl compounds including acetohexamide are effectively reduced by the enzyme. Flavonoids (quercetin and quercitrin) are potent inhibitors for the enzyme, but pyrazole or barbiturates have little effect on the enzyme activity. These findings clearly indicate that the enzyme can be classified as one of the carbonyl reductases. The enzyme also shows both prostaglandin 9-ketoreductase and 3 alpha-hydroxysteroid dehydrogenase activities. Judging from the Kcat/Km values of the enzyme for 4-pyridylketones with a straight-chain alkyl group, a hydrophobic pocket that binds most strongly to a straight-chain alkyl group of five carbon atoms in length appears to be located in the substrate-binding region of the enzyme.     

Benzoyl-coenzyme A reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172

Anoxic metabolism of many aromatic compounds proceeds via the common intermediate benzoyl-CoA. Benzoyl-CoA is dearomatized by benzoyl-CoA reductase (dearomatizing) in a two-electron reduction step, possibly yielding cyclohex-1,5-diene-1-carboxyl-CoA. This process has to overcome a high activation energy and is considered a biological Birch reduction. The central, aromatic-ring-reducing enzyme was investigated for the first time in the denitrifying bacterium Thauera aromatica strain K172. A spectrophotometric assay was developed which was strictly dependent on MgATP, both with cell extract and with purified enzyme. The oxygen-sensitive new enzyme was purified 35-fold with 20% yield under anaerobic conditions in the presence of 0.25 mM dithionite. It had a native molecular mass of approximately 170 kDa and consisted of four subunits a,b,c,d of 48, 45, 38 and 32 kDa. The oligomer composition of the protein most likely is abcd. The ultraviolet/visible spectrum of the protein as isolated, but without dithionite, was characteristic for an iron-sulfur protein with an absorption maximum at 279 nm and a broad shoulder at 390 nm. The estimated molar absorption coefficient at 390 nm was 35,000 M-1 cm-1. Reduction of the enzyme by dithionite resulted in a decrease of absorbance at 390 nm, and the colour turned from greenish-brown to red-brown. The enzyme contained 10.8 +/- 1.5 mol Fe and 10.5 +/- 1.5 mol acid-labile sulfur/mol. Besides zinc (0.5 mol/mol protein) no other metals nor selenium could be detected in significant amounts. The enzyme preparation contained a flavin or flavin-like compound; the estimated content was 0.3 mol/mol enzyme. The enzyme reaction required MgATP and a strong reductant such as Ti(III). The reaction catalyzed is: benzoyl-CoA + 2 Ti(III) + n ATP-->non-aromatic acyl-CoA + 2 Ti(IV) + n ADP + n Pi. The estimated number n of ATP molecules hydrolyzed/two electrons transferred in benzoyl-CoA reduction is 2-4. In the absence of benzoyl-CoA the enzyme exhibited oxygen-sensitive ATPase activity. The enzyme was specific for Mg(2+)-ATP, other nucleoside triphosphates being inactive (< 1%). Mg2+ could be substituted to some extent by Mn2+, Fe2+ and less efficiently by Co2+. Benzoate was not reduced, whereas some fluoro, hydroxy, amino and methyl analogues of the activated benzoic acid were reduced, albeit at much lower rate; the products remain to be identified. The specific activity with reduced methyl viologen as the electron donor was 0.55 mumol min-1 mg-1 corresponding to a catalytic number of 1.6 s-1. The apparent Km values under the assay conditions (0.5 mM for both reduced and oxidized methyl viologen) of benzoyl-CoA and ATP were 15 microM and 0.6 mM, respectively. The enzyme was inactivated by ethylene, bipyridyl and, in higher concentrations, by acetylene. Benzoyl-CoA reductase also catalyzed the ATP-dependent two-electron reduction of hydroxylamine (Km 0.15 mM) and azide. Some of the properties of the enzyme are reminiscent of those of nitrogenase which similarly overcomes the high activation energy for dinitrogen reduction by coupling electron transfer to the hydrolysis of ATP.     

Purification, size, and properties of the complex of orotate phosphoribosyltransferase: orotidylate decarboxylase from mouse Ehrlich ascites carcinoma

Complex U, which contains the last two enzymes (orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.23)) of the six enzymes for the de novo biosynthesis of UMP, has been purified 200-fold from mouse Ehrlich ascites cells. The specific activity of the orotate phosphoribosyltransferase and the orotidylate decarboxylase activities of the complex were 0.115 and 0.290 mumol of product/mg of protein/min; the recovery of the activities was high being 20 to 30%. The rate of the two activities remained similar to that of the homogenate. At the sixth step of the fractionation, one can obtain a fraction that has lost phosphoribosyltransferase activity but retains decarboxylase activity. The apparent molecular weights, as determined by density gradient centrifugation, of the native complex and the fraction containing only decarboxylase activity are identical, 55,700 +/- 4,000. Both activities of complex U are labile to very mild treatments such as dilution, dialysis, or storage at 3 degrees. Dithiothreitol and 5-phosphoribosyl-1-pyrophosphate (PP-Rib-P), but not orotic acid or MgCl2, can stabilize either or both of the enzyme activities. The degree of stabilization by three of these chemicals varies with the reagent(s) used, with the nature of the treatment, and with the concentration of Complex U. When PP-Rib-P, Mg2+ and dithiothreitol are present in the diluting buffer the activity losses were slowed and then followed by a partial recovery of the phosphoribosyltransferase activity. Maximum activities of both enzymes are observed by adding undiluted complex to a complete reaction mixture without preincubation. The complex cannot be exposed to pH values of 4 or below, or pH 9 or above. The stability studies have led to the development of conditions that permit one, for the first time, to subject the complex to electrophoresis and to recover a large percentage of both enzyme activities, rather than only decarboxylase activity as has occurred in the past. The electrophoretic studies indicate that PP-Rib-P produces a complex whose conformation and/or net charge differ significantly from that of the complex in the absence of PP-Rib-P. Kinetic characteristics of the transferase are a pH optimum between 6.5 and 7.5, apparent Km values for orotate, PP-Rib-P, and Mg2+ of 1.9 muM, 16 muM, and 2.9 mM, respectively; for the decarboxylase, a sharp pH optimum of 7.0 is observed, and a Km value for orotidine 5'-phosphate of 0.8 muM.     

Purification and properties of the alpha-acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118

The authors report the case of a very large deterged aortic valve ring abscess detected at long-term after infective endocarditis during investigation of symptomatic consequent aortic regurgitation. The different imaging methods of diagnostic import are reviewed with special emphasis on the role of transoesophageal echocardiography during infective endocarditis.     

GDP-L-fucose pyrophosphorylase. Purification, cDNA cloning, and properties of the enzyme

The enzyme that catalyzes the formation of GDP-L-fucose from GTP and beta-L-fucose-1-phosphate (i.e. GDP-beta-L-fucose pyrophosphorylase, GFPP) was purified about 560-fold from the cytosolic fraction of pig kidney. At this stage, there were still a number of protein bands on SDS gels, but only the 61-kDa band became specifically labeled with the photoaffinity substrate, azido-GDP-L-[32P]fucose. Several peptides from this 61-kDa band were sequenced and these sequences were used for cloning the gene. The cDNA clone yielded high levels of GFPP activity when expressed in myeloma cells and in a baculovirus system, demonstrating that the 61-kDa band is the authentic GFPP. The porcine tissue with highest specific activity for GFPP was kidney, with lung, liver, and pancreas being somewhat lower. GFPP was also found in Chinese hamster ovary, but not Madin-Darby canine kidney cells. Northern analysis showed the mRNA in human spleen, prostate, testis, ovary, small intestine, and colon. GFPP was stable at 4 (o)C in buffer containing 50 mM sucrose, with little loss of activity over a 9-day period. GTP was the best nucleoside triphosphate substrate but significant activity was also observed with ITP and to a lesser extent with ATP. The enzyme was reasonably specific for beta-L-fucose-1-P, but could also utilize alpha-D-arabinose-1-P to produce GDP-alpha-D-arabinose. The product of the reaction with GTP and alpha-L-fucose-1-P was characterized as GDP-beta-L-fucose by a variety of chemical and chromatographic methods.     

Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster)

An analytical procedure was developed to measure bromate residues in baked goods using a sequence of clean-up procedures followed by high performance liquid chromatography (HPLC) with a post-column reaction for oxidants. Deionized water was used to extract bromate from bread samples. The extract was treated with a C-18 solid phase extraction column to remove lipids, a cation exchange column with the silver cation to remove chloride, and an ultrafiltration membrane to remove proteins. Further treatment of the extract with the sodium form of a propylsulphonic acid ion exchange column was necessary to remove the silver that leached from the silver column. The method had a detection limit of 3 ng/g in baked goods. Recoveries of bromate from breads ranged from 73 to 86% at a fortified bromate level of 5-100 ng/g. Pullman-type white bread, produced by a sponge and dough method, was prepared in our laboratory for measurement of residual bromate. The dough was scaled in three different weights at different specific volumes (3.8, 4.1, 4.3), and samples of each of the three weights were baked for six different baking times ranging from 24 to 34 min. When bromate at a level of 25 mg/kg was added to flour, no residual bromate was detected in any of the samples, regardless of weight and baking time.     

Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum

A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized. The estimated molecular mass of the enzyme was 270 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima were 50 degrees C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C. The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate. The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate. Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

Structure and properties of pyruvate decarboxylase and site-directed mutagenesis of the Zymomonas mobilis enzyme

Pyruvate decarboxylase (EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme that catalyzes the penultimate step in alcohol fermentation. The enzyme is widely distributed in plants and fungi but is rare in prokaryotes and absent in animals. Here we review its structure and properties with particular emphasis on how site-directed mutagenesis of the enzyme from Zymomonas mobilis has assisted us to understand the function of critical residues.     

L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12

L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.     

Purification to apparent homogeneity and properties of pig kidney L-fucose kinase

L-Fucokinase was purified to apparent homogeneity from pig kidney cytosol. The molecular mass of the enzyme on a gel filtration column was 440 kDa, whereas on SDS gels a single protein band of 110 kDa was observed. This 110-kDa protein was labeled in a concentration-dependent manner by azido-[32P]ATP, and labeling was inhibited by cold ATP. The 110-kDa protein was subjected to endo-Lys-C digestion, and several peptides were sequenced. These showed very little similarity to other known protein sequences. The enzyme phosphorylated L-fucose using ATP to form beta-L-fucose-1-P. Of many sugars tested, the only other sugar phosphorylated by the purified enzyme was D-arabinose, at about 10% the rate of L-fucose. Many of the properties of the enzyme were determined and are described in this paper. This enzyme is part of a salvage pathway for reutilization of L-fucose and is also a valuable biochemical tool to prepare activated L-fucose derivatives for fucosylation reactions.     

Purification and properties of beta-N-Acetylhexosaminidase from cabbage

beta-N-Acetylhexosaminidase was purified from the extract of cabbage by sequential steps of ammonium sulfate fractionation, chromatofocusing, DEAE-Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 HR gel filtration. By these steps, the purity of the enzyme increased by 256 fold with a recovery of 8%. The purified enzyme was homogeneous as examined by native PAGE. It showed an optimal pH of 4, an optimal temperature of 60 degrees C and a Km of 0.94 mM for hydrolysis of pNp-beta-GlcNAc. The molecular mass of the enzyme determined from filtration through Sephacryl S-200 was 150 kDa. Three subunits with molecular mass of 64, 57 and 51 kDa were observed as determined by SDS-PAGE. NBS (0.025 mM), DEPC (3 mM) and WRK (30 mM) significantly inhibited the activity of the enzyme. The enzyme also showed activity toward pNp-beta-GalNAc, N,N'-diacetylchitobiose, N,N',N"-triacetylchitotriose and N,N',N",N"'-tetraacetyl chitotetraose but showed no activity toward pNp-alpha-GlcNAc, chitin and ethylene glycol chitin.     

Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

Purification, crystallization and properties of triosephosphate isomerase from human skeletal muscle

1. Triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketoisomerase, EC 5.3.1.1) from human skeletal muscle was purified to homogeneity and crystallized. The crystalline enzyme preparation was resolved on polyacrylamide-gel electrophoresis into three isoenzymes. 2. The molecular weight of the enzyme estimated by gel filtration method was found to be 57,400 +/- 3000. Molecular weight determination under dissociation conditions indicated a dimeric subunit structure of the enzyme. 3. The apparent Km for D-glyceraldehyde-3-phosphate as substrate is 0.34 mM, and for dihydroxyacetone phosphate, 0.61 mM. Vmax of the reaction is, respectively, 7200 and 660 units/mg protein at 25 degrees C and pH 7.5. 4. Molecular and kinetic properties of triosephosphate isomerase from human skeletal muscle are very similar to those of rabbit muscle enzyme.     

Purification and properties of beta-galactosidase from Alternaria tenius

beta-Galactosidase from Alternaria tenius was purified to homogeneity from the cultural fluid using acetone precipitation, ion-exchange chromatography on DEAE-cellulose, adsorption on hydroxylapatite and affinity chromatography on N-(beta-D-galactopyranosyl-thiocarbamoyl)-beta-aminocaproyl-AN-Sepharose 4B. The enzyme homogeneity was demonstrated by ultracentrifugation and polyacrylamide gel electrophoresis with SDS or without it. The specific activity of the homogeneous enzyme is 160 u. per mg of protein; mol. weight as determined by various methods is 142 000-176 000, pI = 4.6, temperature optimum is 60-65 degrees, pH optima for o-nitrophenyl-beta-D-galactopyranoside (o-NPG) and lactose are 3.8--4.4 and 3.6--4.8, respectively. The Km values for o-NPG and lactose are 0.21 . 10(-3) and 6.57 . 10-3 M, respectively. The enzyme is a glycoprotein and contains up to 30% of carbohydrates. EDTA and pCMB have no effect on the beta-galactosidase activity. Galactose acts as a competitive inhibitor, while glucose has no inhibiting effect on the enzyme activity.     

Purification, characterization, and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids

The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) was purified 3,000-fold from Streptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 microM for NADPH and 7.7 microM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.     

Purification and properties of filamin, and actin binding protein from chicken gizzard

Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.