Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.     

Rapid purification of two lipase isoenzymes from Candida rugosa

Summary We describe a two-step method for the purification of two lipases (lipases A and B) from C. rugosa. The purification procedure includes Phenyl-Sepharose and Sephacryl HR 100 chromatographies. The enzymes obtained were pure according to criteria of specific activity and neutral sugar content.     

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.     

Purification and Characterization of Glucose 6‐Phosphate Dehydrogenase, 6‐Phosphogluconate Dehydrogenase,and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide,Digoxin, and Dopamine on the Enzymes Activities

The present study was aimed to investigate characterization and purification of glucose–6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'‐ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS‐PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and K_i values with K_m and V_max values of the substrates and coenzymes were determined. According to the obtained K_i and IC₅₀ values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.     

Purification of the intact monomeric 110 kDa form of the androgen receptor from calf uterus to near homogeneity

In the present study, culf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl₂. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [³H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5, while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [³H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa.     

A simple extraction method suitable for PCR-based analysis of plant,fungal, and bacterial DNA

A simple and easy protocol for extracting high-quality DNA from microorganisms and plants is presented. The method involves inactivating proteins by using SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high-molecular-weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous phenol and chloroform, and as many as 100 samples can be processed per day. Absorbency ratios (A₂₆₀/A₂₈₀) of 1.6–2.0 indicated a minimal presence of contaminating metabolites. The DNA was completely digested with 5 restriction enzymes:EcoR I,RsaI,TaqI,EcoR V, andHind III. PCR analysis using enterobacterial repetitive intergenic consensus (ERIC) sequence, sequence-characterized amplified region (SCAR), and random amplified microsatellite (RAMS) primers showed the DNA's compatibility with downstream applications. This procedure is applicable to a range of pathogens and plants and thus may find wide application in quarantine services and marker-assisted selection (MAS) breeding.     

Subcellular Localization of Oxaloacetate Decarboxylase and Its Isolation from Maize Leaves

The activity of oxaloacetate decarboxylase was revealed in leaves of a C₄ plant, maize (Zea mays L.). This activity was unrelated to decarboxylase activities of other enzymes, e.g., NAD-malate dehydrogenase (EC 1.1.1.38) or NADP-malate dehydrogenase (EC 1.1.1.40), and was located in chloroplasts (83.1%). Using a four-step purification procedure, an electrophoretically pure enzyme preparation of oxaloacetate decarboxylase was obtained from maize leaves. The specific activity of the enzyme was 3.150 EU/mg protein, the factor of purification was 40.4, and the yield was 11.0%. The enzyme exhibited Michaelis–Menten kinetics with K _m for oxaloacetate 30 ± 5 M and pH optimum 7.1 ± 0.5. The metabolite-mediated regulation of oxaloacetate decarboxylase activity has been investigated. It is found that sodium chloride (1.0 mM) activates the enzyme, whereas ATP inhibits the enzyme activity.     

The twenty aminoacyl-tRNA synthetases from Escherichia coli. General separation procedure, and comparison of the influence of pH and divalent cations on their catalytic activities

A general separation procedure of the twenty E. coli aminoacyl-tRNA synthetases including either a 105 000 g centrifugation or a polyethyleneglycol-dextran two-phases partition fractionation, and chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite is described. The specific activities of the synthetases have been determined after each chromatographic step and compared to their respective activities in the 105 000 g supernatant. Some aminoacyl-tRNA synthetases were obtained at 80 per cent purity.The presence of phenylmethylsulfonyl fluoride does not significantly modify either the elution patterns of the synthetases during the various chromatographic steps or their specific activities. Thus, contrarily to enzymes from various eukaryotic organisms no significant inactivation of the E. coli aminoacyl-tRNA synthetases occurs via proteolytic processes during the purification procedure.The effects of various factors: pH, magnesium, and other bivalent cations including spermidine, were tested on the aminoacylation and the [³²P] PP_i-ATP isotope-exchange reactions, and the optimal aminoacylation and isotope-exchange conditions determined for 18 of the 20 E. coli aminoacyl-tRNA synthetases.     

Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases

Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C₁₈ columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline. Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC₅₀ in the range of 0.3–2.4 μM. IC₅₀ values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 × 10^–7 M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase. Received: 7 December 1998 / Accepted: 29 March 1999     

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N--CBZ-amino acid-p-nitrophenyl esters, and the highest k _cat/K _m values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.     

Purification of full-length recombinant human and rat type 1 11β-hydroxysteroid dehydrogenases with retained oxidoreductase activities

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11β-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both enzymes were 90–95% pure and exhibited dehydrogenase and reductase activities with K_M values in agreement with those reported in the literature.     

Phosphorylation of the Protein Encoded by the First Open Reading Frame of the MDG4 Transposable Element (gypsy) by Homologous and Heterologous Casein Kinases Type 2

Homogeneous casein kinase type 2 (CK2) was obtained from oocytes of Rana temporaria and cells of Drosophila melanogaster by chromatography on heparin-Sepharose, phosphocellulose, and Mono Q columns using a Pharmacia FPLC system. The procedure was first successfully used for the purification of CK2 from the Drosophila melanogaster cell culture. It has been shown that the protein encoded by the first open reading frame (ORF) of the gypsy transposable element (MDG4) is an effective protein substrate both for homologous and heterologous CK2 from the oocytes of Rana temporaria in vitro. Both enzymes catalyze the incorporation of two moles of phosphate per mole of protein. The K_m and V_max values for the reaction catalyzed by CK2 from the Drosophila cell culture were 32.5 ± 2.1 nM and 70.97 ± 1.89 nmol/min per µg, respectively, and for CK2 from oocytes, these values were 37.6 ± 2.8 nM and 66.02 ± 2.15 nmol/min per µg, respectively.     

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of ketotifen, meloxicam, phenyramidol–HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31?U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2′′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC₅₀ values and K_i constants were 0.012?mM and 0.0008?±?0.00021?mM for ketotifen; 0.029?mM and 0.0061?±?0.00127?mM for meloxicam; 0.99?mM and 0.4340?±?0.0890?mM for phenyramidol–HCl; 138?mM and 28.84?±?4.69?mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol–HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.     

The application of new aminoorganic silanes for affinity chromatography of plant peroxidases on vanillin-liganded controlled porosity glass columns

In this work, new packings consisting of home made controlled porosity glass, new self-prepared aminoorganic silanes and vanillin as a ligand were used for affinity chromatography of some plant peroxidases (donor: H₂O₂ oxidoreductases; EC 1.11.1.7). The procedure allowed simple and relatively high purification of enzymes. The results were compared with those obtained with the material including commercially available γ-aminopropyltriethoxysilane.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

β-N-acetylglucosaminidase and β-galactosidase from aleurone layers of resting wheat grains

We have examined the characteristics of binding to wheat germ agglutinin-Sepharose of β-N-acetylglucosaminidase and β-galactosidase from aleurone layers of resting wheat grains. Although the enzymes interacting with wheat germ agglutinin-Sepharose could be extracted by a procedure which did not involve any solubilizing treatments, the highest activity of these enzymes was obtained by extracting and sonicating the tissues in the presence of 0.5% Triton X-100. The pH optimum and time-course of binding as well as the effect of some divalent ions on the binding were studied. The largest part of the bound enzymes was eluted at low concentration of N-acetyl-D-glucosamine (0.05 M), although smaller amounts were still eluted at higher molarities (0.1 and 0.2 M). D-Mannose, D-glucose and L-fucose failed to replace N-acetyl-D-glucosamine in eluting the enzymes bound to wheat germ agglutinin-Sepharose, whereas N-acetyl-D-galactosamine was much less effective than N-acetyl-D-glucosamine. The catalytic properties of the enzymes remained unchanged after the binding to wheat germ agglutinin-Sepharose, although the K_m values of the free and lectin-bound enzymes were slightly different. A rapid and easy three-step procedure of purification, mainly based on affinity chromatography on wheat germ agglutinin-Sepharose, is described. It allows purification of β-galactosidase and β-N-acetylglucosaminidase over 200-fold. β-N-Acetylglucosaminidase has been further purified to electrophoretic homogeneity and also characterized.     

Extractive partition of L-aspartase and fumarase fromEscherichia coli using aqueous polyethylene glycol-ammonium sulfate systems

Summary Inorganic sulfate salts are used to form aqueous two-phase systems with polyethylene glycol (PEG) for enzyme purification. Two enzymes, L-aspartase and fumarase produced byEscherichia coli are efficiently separated into different phases in spite of the high degree of similarity in molecular weight and amino acid sequence between them. The ratio of L-aspartase to fumarase in the PEG-rich phase is more than sixty (60) times the ratio before extraction. A high degree of purification in a single extraction step can be achieved by careful selections of PEG molecular weight, pH, cation of the salts, and sodium chloride levels. Cations of sulfate-containing salts in the following order: NH ₄ ⁺ >Na⁺>Mg²⁺ tend to increase the partition of L-aspartase in the PEG-rich phase. The maximal degree of enzyme purification is obtained by using PEG 4000 and ammonium sulfate as a phase system at a stable pH for both enzymes.     

Cloning of the<Emphasis Type="Italic"> pelA</Emphasis> gene from<Emphasis Type="Italic"> Bacillus licheniformis</Emphasis> 14A and biochemical characterization of recombinant,thermostable, high-alkaline pectate lyase

The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K _m and V _max of the fusion protein were 0.56 g/l and 51 µmol/min, respectively. The activity of purified PelA^His was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.     

The effect of homologous amino acid replacement on the conformation of oligopeptides. I. Synthesis of co-oligopeptides containing methionine and valine

The preparation of a series of co-oligopeptides Boc-Val-Met_n-OMe(n = 1–6), as sell as Boc-Met₃-Val-et₂-OMe and Boc-Met₃-Val-Met₃-Ome, is described. The synthesis was carried out by a classic method employing the mixed anhydride procedure (isobutylchloroformate) for all coupling reactions. All oligopeptides after purification were homogenous on silica thin layers and gave correct elemental analysis. They were judged to be optically pure using molar rotation studies at the sodium D line.