Deoxyhypusine hydroxylase from rat testis. Partial purification and characterization

Deoxyhypusine hydroxylase, the enzyme that catalyzes the formation of hypusine from deoxyhypusine in eukaryotic initiation factor 4D, has been partially purified from rat testis. The partially purified enzyme requires only the addition of certain sulfhydryl compounds for catalytic activity, dithiothreitol being the most effective. Its lack of dependency on the alpha-keto acid-dependent dioxygenase cofactors, Fe2+, alpha-ketoglutarate, and ascorbic acid, its failure to decarboxylate stoichiometrically alpha-ketoglutarate with deoxyhypusine hydroxylation, and its strong and specific inhibition by Fe2+ all suggest a catalytic mechanism of this enzyme unlike that of the prolyl and lysyl hydroxylases.     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶羟基化酶组分的纯化和性质

Methylomonassp．GYJ3菌株中经DEAE-SepharoseCL-6B阴离子交换层析和SephacrylS300凝胶层析分离纯化出甲烷加氧酶羟基化酶组分．经HPLC分析,纯度大于90％,分子量为240kD,纯化倍数为3．9,比活为225nmol环氧丙烷每分钟毫克蛋白．SDS-PAGE表明,羟基化酶由三个亚基组成,亚基分子量为56、43、27kD．ICPAES测定羟基化酶的Fe含量为2.1molFe每摩尔蛋白．HPLC法用于甲烷单加氧酶羟基化酶组分的纯化,纯化的羟基化酶组分比活为541nmol（环氧丙烷）每分钟毫克蛋白,是两步LC法纯化的羟基化酶的两倍,Fe含量为3．78molFe每摩尔蛋白．催化性质研究表明羟基化酶能够被化学还原剂还原为还原态羟基化酶,还原态的羟基化酶单独存在时表现出MMO活性,说明它是MMO活性中心,天然态的羟基化酶单独存在时无MMO活性,加入粗酶液中MMO活性明显增加,说明GYJ3菌中MMO是一个复合酶系．     

Purification and characterization of D-3-aminoisobutyrate-pyruvate aminotransferase from rat liver

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) was purified 1900-fold from rat liver extract. The purified enzyme showed a molecular mass of 180 kDa by gel-permeation HPLC analysis using a TSK gel G3000SW column. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in identification of a single band of approx. 50 kDa, indicating that the native enzyme is probably a tetrametric protein. The specific activity of the purified enzyme was 1.14 mumol/min per mg protein. D-3-Aminoisobutyrate and beta-alanine were good amino donors. The Km value for L-3-aminoisobutyrate was 100-times larger than that for the D-isomer. The apparent Km values for D-3-aminoisobutyrate and beta-alanine were 35 and 282 microM, respectively. Pyruvate, glyoxylate, oxalacetate, 2-oxo-n-valerate, and 2-oxo-n-butyrate were good amino acceptors. The apparent Km values for pyruvate and glyoxylate were 32 and 44 microM, respectively.     

Purification and initial characterization of deacetoxycephalosporin C synthase from Streptomyces clavuligerus

Deacetoxycephalosporin C synthase, the penicillin N ring expansion enzyme from Streptomyces clavuligerus, was purified to near homogeneity, as judged by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The synthase was monofunctional and could be completely separated from deacetoxycephalosporin C hydroxylase activity early in the purification sequence. Synthase specific activity was increased 97-fold over crude cell-free extracts, and the purified enzyme appeared to be a monomer with a molecular weight of 36,000 and a Km for the penicillin N substrate of 50 microM. Deacetoxycephalosporin C synthase activity required alpha-ketoglutarate, Fe2+, and oxygen and was specifically stimulated by ascorbate and dithiothreitol. The enzyme was sensitive to thiol-specific inhibitors, the most effective of which was N-ethylmaleimide.     

Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase

Methylmalonate semialdehyde dehydrogenase was purified from rat liver in order to define the distal portion of valine catabolism and related pathways in mammals. The purified enzyme is active with malonate semialdehyde and consumes both stereoisomers of methylmalonate semialdehyde, implicating a single semialdehyde dehydrogenase in the catabolism of valine, thymine, and compounds catabolized by way of beta-alanine. The oxidation of malonate and methylmalonate semialdehydes by this enzyme is CoA-dependent, the products being acetyl-CoA and propionyl-CoA, respectively. Expected activity with ethylmalonate semialdehyde as substrate was not found. Methylmalonate semialdehyde dehydrogenase was separated on DEAE-Sephacel into two isoforms which differ in mobility during nondenaturing polyacrylamide gel electrophoresis. The two forms are immunologically cross-reactive and exhibit the same N-terminal sequence, suggesting that one form is the product of the other. The monomer molecular mass, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58 kDa. The native molecular mass, estimated by gel filtration, was 250 kDa, suggesting a tetrameric structure.     

Purification and partial characterization of the hydroxylase component of the methanesulfonic acid mono-oxygenase from methylosulfonomonas methylovora strain M2.

The reductase enzyme and the hydroxylase enzyme of the three-component methanesulfonic acid mono-oxygenase (MSAMO) from Methylosulfonomonas methylovora were purified. Purification of the reductase from M. methylovora using a range of chromatographic techniques was accompanied by complete loss of activity. Expression of the reductase as a glutathionine S-transferase fusion protein in Escherichia coli cells was successful as judged from the size of the polypeptide band obtained on induction with isopropyl thio-beta-D-galactoside. Subsequent affinity purification of the fusion protein, however, led to a protein extract containing only glutathionine S-transferase protein, indicating that the fusion protein was unstable in vitro. The hydroxylase component of the MSAMO was purified from M. methylovora to near electrophoretic homogeneity using Q-Sepharose, hydroxyapatite and Mono Q chromatography. SDS/PAGE of the purified hydroxylase showed a single band at approximately 43.7 kDa for the alpha-subunit and a double band at approximately 23 kDa for the beta-subunit. MS scans obtained with matrix-assisted laser desorption/ionization and electrospray ionization showed single peaks for both subunits, with a mass of 48 145.4 Da for alpha, 20 479.1 Da for beta, and 68 624.5 for the alphabeta-monomer. Gel filtration revealed a mass of 209 kDa, suggesting an alpha3beta3 structure for the native enzyme. Purified hydroxylase enzyme exhibited absorbance maxima at 330 nm, 460 nm and 570 nm, indicating the presence of iron-sulfur centres. The protein preparations contained 1 mol sulfide and 3-4 mol iron per mol alphabeta-monomer. Chromium, cobalt, copper, lead, nickel, molybdenum, tungsten and vanadium were not found. Flavins were also absent. Antibodies raised against the native hydroxylase enzyme cross-reacted with cell-free extract from M. methylovora cells grown with methanesulfonate, but not with extract from cells grown with methanol, confirming that MSAMO was specifically induced during growth on methanesulfonate.     

Purification and characterization of an oxygen-stable carbon monoxide dehydrogenase of Methanothrix soehngenii

Carbon monoxide dehydrogenase was purified to apparent homogeneity from Methanothrix soehngenii. In contrast with the carbon monoxide dehydrogenases from most other anaerobic bacteria, the purified enzyme of Methanothrix soehngenii was remarkably stable towards oxygen and it was only slightly inhibited by cyanide. The native molecular mass of the carbon monoxide dehydrogenase of Methanothrix soehngenii determined by gel filtration was 190 kDa. The enzyme is composed of subunits with molecular mass of 79.4 kDa and 19.4 kDa in an alpha 2 beta 2 oligomeric structure. The enzyme contains 1.9 +/- 0.2 (n = 3) mol Ni/mol and 19 +/- 3 (n = 3) mol Fe/mol and it constitutes 4% of the soluble cell protein. Analysis of enzyme kinetic properties revealed a Km of 0.7 mM for CO and of 65 microM for methyl viologen. At the optimum pH of 9.0 the Vmax was 140 mumol of CO oxidized min-1 mg protein-1. The enzyme showed a high degree of thermostability.     

Purification and characterization of an NADH oxidase from Eubacterium ramulus

An NADH oxidase from the strictly anaerobic Eubacterium ramuluswas purified to homogeneity. The enzyme is composed of two types of subunits with molecular masses of 40 and 30 kDa. The molecular mass of the native enzyme is 450 kDa according to gel filtration and PAGE analysis. Six to eight mol of FAD were found per mol of native enzyme. The NADH-specific enzyme was inhibited by N-bromosuccinimide and sulfhydryl reagents such as N-ethylmaleimide, CuCl(2) or ZnCl(2). The physiological function of the purified enzyme is unclear, but the demonstration of NADH-dependent O(2)-consumption suggests that it plays a role in the scavenging of oxygen.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.     

Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174

An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH(inf2)-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Se r-Gln-Pro-Gly.     

Purification and characterization of an invertase produced by Aspergillus ochraceus TS.

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.     

Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.     

Purification and properties of invertase from the flowers of Woodfordia fruticosa

Invertase (beta-fructofuranosidase, EC 3.2.1.26) was purified from the flowers of Woodfordia fruticosa, which is used to prepare certain fermented Ayurvedic drugs. The enzyme was purified to near homogeneity as judged by native PAGE with a yield of 10.7%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.8 and 4.42. The molecular mass of the purified enzyme as determined by elution through Sepharose 4B gel column was found to be approximately 280 kDa. SDS-PAGE of the purified enzyme showed that the enzyme is composed of three subunits with molecular mass of 66, 43 and 40 kDa. The enzyme showed a broad pH optimum between 4.0-7.0. Optimum assay temperature was 37 degrees C and above 45 degrees C, the enzyme activity slowly declined and inactivated around 80 degrees C. The apparent Km value of the enzyme for sucrose was 160 mM.     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Purification and properties of bromoperoxidase from Pseudomonas pyrrocinia

A bromoperoxidase was purified and partially characterized from Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. The purified enzyme preparation was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular mass of the enzyme was estimated to be 154 kDa +/- 3 kDa as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band with the mobility of a 76-kDa species. Therefore, in solution at neutral pH, bromoperoxidase exists as a dimeric species. The isoelectric point was 5.0. The prosthetic group of this procaryotic bromoperoxidase was ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. The purified enzyme showed brominating as well as peroxidase and catalase activity.     

Purification and characterization from rat kidney membranes of a novel platelet-activating factor (PAF)-dependent transacetylase that catalyzes the hydrolysis of PAF, formation of PAF analogs, and C2-ceramide

We have previously identified two enzyme activities that transfer the acetyl group from platelet-activating factor (PAF) in a CoA-independent manner to lysoplasmalogen or sphingosine in HL-60 cells, endothelial cells, and a variety of rat tissues. These were termed as PAF:lysoplasmalogen (lysophospholipid) transacetylase and PAF:sphingosine transacetylase, respectively. In the present study, we have solubilized and purified this PAF-dependent transacetylase 13,700-fold from rat kidney membranes (mitochondrial plus microsomal membranes) based on the PAF:lysoplasmalogen transacetylase activity. The mitochondria and microsomes were prepared and washed three times, then solubilized with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. The solubilized fractions from mitochondria and microsomes were combined and subjected to sequential column chromatographies on DEAE-Sepharose, hydroxyapatite, phenyl-Sepharose, and chromatofocusing. The enzyme was further purified by native-polyacrylamide gel electrophoresis (PAGE) and affinity gel matrix in which the competitive inhibitor of the enzyme, 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphoethanolamine was covalently attached to the CH-Sepharose. On SDS-PAGE, the purified enzyme showed a single homogeneous band with an apparent molecular mass of 40 kDa. The purified enzyme catalyzed transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing N-acetylsphingosine (C2-ceramide). In addition, this enzyme acted as a PAF-acetylhydrolase in the absence of lipid acceptor molecules. These results suggest that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators.     

Purification and characterization of alpha-keto amide reductase from Saccharomyces cerevisiae

An NADPH-dependent alpha-keto amide reductase was purified from Saccharomyces cerevisiae. The molecular mass of the native enzyme was estimated to be 33 and 36 kDa by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The purified enzyme showed a reducing activity not only for aromatic alpha-keto amides but also for aliphatic and aromatic alpha-keto esters. The internal sequence of the enzyme was identical with that of a hypothetical protein (ORF YDL 124w) coded by yeast chromosome IV.     

Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain

We have purified S-adenosylmethionine (AdoMet) synthetase about 3000-fold from bovine brain extract. The Km values of the enzyme for L-methionine and ATP were 10 and 50 microM, respectively. An apparent molecular mass of the enzyme was estimated to be 160 kDa by gel filtration on a Sephacryl S-200 column. Sucrose density gradient centrifugation gave a sedimentation coefficient of 8 S. Polyacrylamide gel electrophoresis of the purified enzyme in native system revealed a single protein band, whereas two polypeptide bands with molecular masses of 48 kDa (p48) and 38 kDa (p38) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. Antibody against bovine brain AdoMet synthetase was prepared by injecting the purified enzyme into a rabbit. Immunoblot analysis revealed that the antibody recognized both p48 and p38 in the impure enzyme preparations from bovine brain as well as in the purified enzyme. Specific antibodies against p48 and p38 were separated from the immunoglobulin fraction by an affinity purification, both of which inhibited the enzyme activity. These results indicate that AdoMet synthetase from bovine brain consists of two different polypeptides, p48 and p38.