Sea anemone collagen: Further evidence for the existence of only oneα-chain type

Summary Collagen from an inferior invertebrate, namely from the sea anemoneActinia equina L., appears to consist of three identical subunits (-chains). New evidence for this situation is reported on the basis of preparative-scale experiments.No fractionation of sea anemone collagen was obtained by CM-cellulose chromatography which is the method of choice for the isolation of vertebrate collagen subunits. On the other hand, the-component (mol. wt., 95.000) could be isolated by gel chromatography. It was homogenous when investigated by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulphate) and by ion exchange chromatography on CM-cellulose. The amino acid composition of the-component was identical with that of unfractionated collagen.Divergent evolution of the collagen molecule appears likely since vertebrate collagen molecules are made up of different-chain types.     

Antifungal activity of some discomycetes

An antifungal glycoprotein compound was obtained from the culture filtrate of Ciboria rufo-fusca, by ultrafiltration and fractionation through D.E.A.E. cellulose and ultrogel Ac A34 chromatography. The purified material was homogeneous on polyacrylamide gel electrophoresis and was sensitive to the action of glucuronidase and catalase, and partly denatured by urea.The antifungal properties of the discomycete: Ciboria rufo-fusca was described in the first part of this work (6). The second part deals with the nature of the antifungal substance produced by Ciboria rufo-fusca.     

Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences

Summary Type II DNA topoisomerase has been isolated from inflorescences of cauliflower (Brassica oleracea var. botrytis) through a sequence of polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex, hydroxyapatite and phosphocellulose. The molecular weight of the native enzyme, based on sedimentation coefficient (9S) and gel filtration analysis (Stokes radius, 60 Å), was estimated to be 223 000. This enzyme was able to catalyze fully the relaxation of supercoiled DNA by breaking and then rejoining the double-stranded DNA. The breaking reaction was reversible by a change in salt concentrations. When an antitumor drug, 4-(9-acridinylamino)-methanesulfon-m-anisidide, was added to the topoisomerase reaction, DNA cleavage fragments were accumulated; and this suggested that the drug interfered with the reaction at the rejoining step. This enzyme also catalyzed the formation of DNA catenanes in the presence of 8% polyethylene glycol or histone H1, while few catenanes were formed in the presence of spermidine, which was highly effective on a bacterial enzyme.     

Fractionation of DNA from Bacillus subtilis and its transforming activity for various markers

The physical, chemical, and functional heterogeneity of tranforming DNA was studied by preparative fractionation techniques providing resolution with respect to differences in molecular weight (gel filtration, sucrose density gradient), base composition (CsCl density gradient), or both these parameters simultaneously (methylalbumin-coated celite 545 MAK column). A comparison of the basic characteristics of the obtained fractions (melting temperature, T _m; density, ; sedimentation coefficient, S _20,w; and transforming activity for ade, leu, and met markers) showed that the factor decisive for functional activity represents, in addition to the sequential arrangement of nucleotides in the chain, the average base composition. Hence, using the methylalbumin column or CsCl density gradient centrifugation, DNA fractions can be isolated which show a several times higher transforming activity for any of the markers examined. By contrast, the remaining fractionation methods, even though considerably decreasing the heterogeneity of the fractions as regards their molecular weight (such as zonal centrifugation), do not offer a possibility of fractionation of the activity for individual markers. This indicates a statistically random degradation of transforming DNA during its isolation. The order of the investigated markers according to their guanine-cytosine content is ade leu met and corresponds also to the order of their positions on the genetic map of Bacillus subtilis.     

Purification and Characterization of an Extracellular Non-Aspartyl Acid Protease (pumAe) from <Emphasis Type="Italic">Ustilago maydis</Emphasis>

The proteinase pumAe was purified to homogeneity from haploid U. maydis FB1 growing on acid mineral medium. The purification procedure consisted of ammonium sulfate fractionation and gel filtration chromatography, resulting in a 7.7% recovery and a 15.1-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 72 kDa and 74 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 4.0 and at 45°C toward hemoglobin, and the pI was determined to be 5.5. The effects of six protease inhibitors on pumAe were tested, and no inhibitory effect was observed. The pure enzyme degraded gelatin and albumin, but casein and collagen were not degraded. The K_m value was 3.5 M, and the V_max value was 11430 mol h^–1 mg^–1 for Suc-R-P-F-H-L-L-V-Y-MCA.     

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 g–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.     

Extraction of RAPD-friendly DNA from Laminaria japonica (Phaeophyta) after enzymatic dissociation of the frozen sporophyte tissues

A new extraction protocol has been developed to obtain high quality DNAfrom Laminaria japonica, which involves enzymatic dissociation ofsporophyte tissues and subsequent elimination of the remainingpolysaccharides with cetyltrimethyl ammonium bromide. Unicells isolatedfrom frozen kelp tissues with alginate lyase prepared from the abalone Haliotis diversicolor were used to extract total DNA; the yield wasapproximately 13 to 22.5 g DNA g^-1 (wet sporophyteweight). The average size of genomic DNA was around 23 kb estimated byagarose gel electrophoresis, and the purity of total DNA determinedspectrophotometrically as the ratio of OD₂₆₀/OD₂₈₀ wasabout 1.7. The extracted kelp DNA (20–40 ng) could be usedsuccessfully as a template for polymerase chain reaction (PCR) under theoptimized conditions (100 M dNTP, 0.2 M primer, 1.0unit Taq DNA polymerase). The random amplified polymorphicDNA (RAPD) patterns were highly reproducible. These results suggest thatthe contamination by soluble polysaccharides which interferes with RAPDreproducibility was largely controlled. This RAPD-suited method for DNAextraction from kelp sporophytes using enzyme treatment providedsufficient material, and was inexpensive and convenient to carry out.     

Purification and Some Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

A β-xylosidase of a thermophilic fungus, Malbranchea pulchella var. sulfurea No. 48, was purified 99-fold from the culture filtrate after ammonium sulfate fractionation, DEAE-cellulose column chromatography, column electrophoresis and gel filtration on Sephadex G–200. The purified enzyme was found to be homogeneous upon ultracentrifugal analysis, disc electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be 26,000 by gel filtration, and the sedimentation coefficient was calculated to be 2.78S. at 280 nm in phosphate buffer (pH 6.7) was 13.2. The optimum pH was found to be in the range of 6.2~6.8, and the optimum temperature was 50°C.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Purification of ACV synthetase fromStreptomyces clavuligerus

Summary By a combination of protamine sulfate treatment, ammonium sulfate fractionation, gel filtration and hydrophobic interaction chromatography, an active -(L--aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from the prokaryoteStreptomyces clavuligerus was purified 135-fold to give a single major protein band on SDS-PAGE. Its size appears to be approximately 360 kDa which is very similar to that of the enzyme from the eukaryote,Cephalosporium acremonium.     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Use of thin layer chromatography for detection and high performance liquid chromatography for quantitating gliotoxin from rice cultures ofAspergillus fumigatus Fresenius

Gliotoxin, a mycotoxin with antimicrobial and immunosuppressive capabilities, is produced by several genera of fungi including the pathogenic fungusAspergillus fumigatus. The ability of selected isolates ofA. fumigatus to produce gliotoxin on three different media was tested and a thin layer chromatographic and high performance liquid chromatographic method for quantitation of gliotoxin from rice culture was developed and is described. Rice cultures were extracted with chloroform and the resulting extract was partially purified by precipitation with petroleum ether and cleanup by gel permeation chromatography. Gliotoxin was detected by thin layer chromatography and quantitated by high performance liquid chromatography using a U.V. absorbance detector with a 254 nm filter and a mobile phase of methanol-water 4357 (V/V) with a flow rate of 2.0 ml/min. The retention time for gliotoxin was approximately 4.8 min. From rice samples spiked with gliotoxin concentrations of 0.67, 1.33, 2.67, 4.00 and 5.33g/g the average recovery was 83.8%.     

Purification and Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH₄)₂SO₄ fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g^–1 s^–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b ₆ and an internal sequence of polyphenol oxidase.Abbreviations DMSO dimethylsulfoxid - HIC hydrophobic interaction chromatography - MGDG monogalactosyl diacylglycerol - VDE violaxanthin deepoxidase A preliminary report of these results was presented at the Xth Int. Congress on Photosynthesis, Montpellier, France, 1995.     

Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12

A novel fibrinolytic enzyme from Rhizopus chinensis 12 was purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange, and gel filtration chromatography. The purification protocol resulted in a 893-fold purification of the enzyme, with a final yield of 42.6%. The apparent molecular weight of the enzyme was 18.0 kDa, determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 16.6 kDa by gel filtration chromatography, which revealed a monomeric form of the enzyme. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5±0.1. The enzyme hydrolyzed fibrin. It cleaved the , , and chains of fibrinogen simultaneously, and it also hydrolyzed casein and N-succinyl-Ala-Ala-Pro-Phe-pNA. The enzyme had an optimal temperature of 45°C, and an optimal pH of 10.5. EDTA, PCMB, and PMSF inhibited the activity of the enzyme, and SBTI, Lys, TPCK, and Aprotinine had no obvious inhibition, which suggested that the activity center of the enzyme had hydrosulfuryl and metal. The first 12 amino acids of the N-terminal sequence of the enzyme were S-V-S-E-I-Q-L-M-H-N-L-G and had no homology with that of other fibrinolytic enzyme from other microbes.     

Identification of Arsenobetaine as a Major Arsenic Compound in the Ivory Shell Buccinum striatissimum

A restriction endonuclease, designated as DmaI, was purified from cell-free extracts of Deleya marina IAM 14114 by streptomycin treatment, ammonium sulfate fractionation and two steps of chromatographies on heparin-Sepharose CL-6B and Mono Q (HR 5/5, FPLC). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis and a ligation-recutting test. The relative molecular mass measurements of the purified enzyme gave 28,000 daltons by SDS-polyacrylamide gel disk electrophoresis and 56,000 daltons by gel filtration. These data indicated that the purified enzyme (56,000 daltons) has a dimeric structure composed of two 28,000-dalton subunits. The isoelectric point was 5.5. The purified enzyme worked best at 37°C in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris–HCl, 7 mm 2-mercaptoethanol, 7 mm MgCl₂ and 100 mm NaCl (pH 7.5). The enzyme was stable up to 55°C and between pH 7.0 and 9.0. The purified enzyme recognizes the palindromic hexanucleotide DNA sequence 5′-CAGCTG-3′, cuts between G and C and produces a flush end (isoschizomer of PvuII).     

Rapid,High Quality DNA Isolation from Cypress (Cupressus sempervirens L.) Needles and Optimization of the RAPD Marker Technique

A protocol for extracting high quality DNA from cypress (Cupressus sempervirens L.), a gymnosperm of economic and ecological importance to the Mediterranean, is presented using the commercially supplied DNeasy^TM Plant Mini Kit (QIAGEN GmbH, Germany). Additional steps were introduced in the QIAGEN protocol to significantly enhance DNA quantity and quality. For one sample, the procedure can be completed in less than one hour, and more than 10 samples can be processed in a day. DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The average DNA yield obtained from 100 mg starting material was around 22 g, which compares very favorably with numbers indicated by the manufacturer. Additionally, restriction and PCR analyses of the extracted DNA showed its compatibility with downstream applications. Using this DNA, the parameters for the randomly amplified polymorphic DNA (RAPD) protocol were optimized based on the use of (1) an AmpliTaq^® DNA Polymerase, Stoffel fragment (Perkin-Elmer), and (2) a high initial denaturation temperature (97.5 °C). Reproducible amplification products were achieved in all PCR reactions. Seven to eight bands per primer were obtained with most individuals. This represents a number at the high end of published results with other plant species.     

Purification and Properties of an Aminopeptidase from a Protamine-degrading Marine Bacterium

A protamine-degrading marine bacterium was isolated from marine soil and identified as Aevomonas salmonicida subsp. based on its taxonomical characteristics. An alanine-specific aminopeptidase, called aminopeptidase K, from an extract of the strain was purified and characterized. The aminopeptidase K was purified about 80-fold by fractionation with ammonium sulfate and column chromatography on QA-52 cellulose, Phenyl Superose and Superose 12. The purified enzyme is composed of 6 subunits of 86 kDa with a molecular mass of 520 kDa according to gel filtration and SDS–PAGE. The N-terminal sequence of the enzyme was H · Gly-Gln-GIn-Pro-Gln-Ile-Lys-Try-Tyr-His-Asp-Tyr-Asp-Ala-Pro-Asp-Tyr-Tyr-Ile-Thr-. It is inhibited by monoiodoacetate, N-ethylmaleimide, and puromycin. The Michaelis constant (K_m) and the maximal rate of hydrolysis (V_max) were, respectively, 0.28 mm and 49.4 μmol/min/mg for the l-Ala-β-naphthylamide substrate. The optimum pH and optimum temperature were 6.5 and 45°C, respectively. The purified enzyme was highly specific to l-Ala-β-naphthylamide.     

Role of bean lectins inRhizobium phaseoli-Phaseolus vulgaris interactions. Some properties of lectins from two bean cultivars

Lectins from twoPhaseolus vulgaris L. cultivars were isolated and purified by salt fractionation, affinity chromatography and gel permeation chromatography. The cultivars used were: alubia, with a low-nodulating ability, and Bat 76 with a good symbiotic aptitude. Differences in properties of the two lectins were noted: alubia lectin gave only one peak with haemagglutinating activity following gel permeation chromatography while Bat 76 yielded two active peaks, although both lectins had several bands of about 30 kDa following gel electrophoresis, Bat 76 lecting had three bands of about 50 kDa which were not present in alubia and red kidney bean lectins. Peptide-mapping, by limited proteolysis and two dimensional gel electrophoresis, also showed differences between the lectins which are therefore judged to be different.     

Adenosine deaminase from Azotobacter vinelandii. Purification and properties

Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65°C, fractionation with ammonium sulfate, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Purified adenosine deaminase was effectively stabilized by the addition of ethylene glycol. The molecular weight of the enzyme was estimated to be 66,000 by gel filtration on Sephadex G-150. The enzyme specifically attacked adenosine and 2-deoxyadenosine to the same extent, and formycin A to a lesser extent. The pH optimum of the enzyme was observed at pH 7.2. Double reciprocal plot of initial velocity versus adenosine concentration was concave upward, and Hill interaction coefficient was calculated to be 1.5, suggesting the allosteric binding of the substrate. ATP inhibited adenosine deaminase in an allosteric manner, whereas other nucleotides were without effect. The physiological significance of the enzyme was discussed in relation to salvage pathway of purine nucleotides.