Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor

The technique of three-phase partitioning (TPP) was used to purify a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana). This process of purification is a potential method used for separation of proteins directly from large volumes of crude suspension. It involves the addition of a salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (t-butanol). The addition of t-butanol, in the presence of ammonium sulphate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The process was carried out in two steps. The various conditions required for attaining efficient purification of the protein fractions were optimized. It was seen that 30% ammonium sulphate saturation with 1:1 ratio of crude extract to tert-butanol gave 8.9- and 8.65-fold purification with 83% and 80% yield of amylase inhibitor and trypsin inhibitor, respectively, in step I. In TPP-step II, 60% ammonium sulphate saturation and ratio of aqueous phase to t-butanol of 1:2 gave maximum 20.1- and 16-fold purification with 39.5% and 32% yield of amylase inhibitor and trypsin inhibitor, respectively. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the inhibitor protein showed substantial purification and the molecular weight of the protein was found to be 14 kDa.     

A rapid method for the isolation of coagulation factor XII from human plasma

Zinc chelate affinity chromatography was used to develop a rapid, three-step procedure to isolate coagulation factor XII from human plasma. The first step was ammonium sulphate fractionction which gave a 2-fold purification and 90% recovery in the 25–50% saturation fraction. The second step was zinc chelate affinity chromatography which gave a 240-fold purification and 67.5% recovery. The third step was zinc chelate affinity chromatography again, but with the application of a pH gradient. The overall recovery of zymogen factor XII was 21.7% and the total purification was 1992-fold. The purified factor XII had an apparent molecular weight of 77 600 as determined by SDS-polyacrylamide gel electrophoresis and a specific activity of 50 units/mg on a clotting assay.     

Trypsin inhibitor from bambara pea

An antitryptic activity has been identified in the flour of dry non-germinated seed of an African leguminous plant, the bambara pea (Voandzeia subterranea). This inhibitor has been purified by trichloracetic acid and ammonium sulphate precipitations followed by gel filtration on Sephadex G25, ion exchange chromatography and gel filtration on Sephadex G75. Antitryptic activity increased 50-fold. Its purity has been verified by electrophoresis on polyacrylamide gel and gel chromatography. Its MW is 13 200 in the denatured and reduced forms and 26 300 in the native form. It is resistant to thermal denaturation and appears to be in monomeric form when entirely denatured.     

Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet

An H⁺-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.     

Improved purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD⁺/2 mM Na₂SO₃). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).     

Purification of a special estrogen-binding protein from the liver of rats by affinity chromatography on estradiol sepharose

A procedure has been developed for purification of a special rat liver estrogen-binding protein. It includes protein precipitation by ammonium sulfate, gel filtration, ion exchange chromatography, and affinity chromatography on estradiol sepharose. The protein is purified 2260-fold with a 27% yield. Upon electrophoresis in 10% PAG in the presence of sodium dodecylsulfate the protein gives one polypeptide strip with a molecular weight of 31.200 +/- 400 dalton.     

Purification and properties of aspartate transcarbamylase from Mycobacterium smegmatis

Aspartate transcarbamylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) has been purified from Mycobacterium smegmatis TMC 1546 using streptomycin sulphate precipitation, ammonium sulphate precipitation, DE-52 chromatography, second ammonium sulphate precipitation, Sephadex G-200 gel filtration, and aspartate-linked CNBr-activated Sepharose 4B affinity chromatography in successive order. The enzyme was purified 231.6-fold, and the preparation was found to be homogeneous on column chromatography and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 246,000 and was composed of two asymmetrical subunits. The kinetic and regulatory properties of aspartate transcarbamylase from M. smegmatis were also studied. The enzyme was found to be an allosteric in nature with carbamyl phosphate showing positive cooperativity and UMP exhibiting a negative cooperativity. CTP was found to be the most potent inhibitor among nucleotides. Phosphate acted as a non-competitive product inhibitor with respect to aspartate. Succinate and maleate exerted a competitive inhibition when aspartate was the variable substrate.     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.     

Purification and some properties of isocitrate dehydrogenase from Paracoccus denitrificans

NADP-dependent isocitrate dehydrogenase (ICDH) from the bacterium Paracoccus denitrificans was purified to homogeneity. The purification procedure involved ammonium sulphate fractionation, ion exchange chromatography, and gel permeation chromatography. The specific activity of purified ICDH was 801 nkat/mg, the yield of the enzyme 58%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. ICDH is a dimer composed of two probably identical subunits of relative molecular weight 90,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 5.6; the presence of Mn2+ is essential for enzyme activity. The absorption and fluorescence spectra of the homogeneous enzyme were measured as well.     

Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus

A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B-hygromycin-B. The combined active fractions showed a single protein band (41 kDa) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 microM for hygromycin B and ATP, respectively, were deduced.     

Purification and characterization of an extracellular amylase from Lactobacillus plantarum strain A6

Extracellular amylase from Lactobacillus plantarum A6 was purified by fractionated precipitation with ammonium sulphate and by anion exchange chromatography. The homogeneity of the purified fraction was tested by polyacrylamide gel electrophoresis and showed multiple amylase forms. A major form had an estimated molecular weight of 50 kDa. It was identified as an α-amylase, with an optimum pH of 5.5, an optimum temperature of 65°C and K _m value of 2.38 g l^-1 with soluble starch substrate. The enzyme was inhibited by N -bromosuccinimide, iodine and acetic acid. The enzyme activation energy was 30.9 kJ mol^-1.     

Isolation of a histone-specific proteinase from brain cell nuclei of rats]

A method for isolating histone proteinase of rat brain chromatin is described, including ammonium sulphate fractionation gel filtration on sefacryl 5200, ion exchange chromatography on mono S and affinity chromatography with benzamidine sefarose. The enzyme molecular weight equals 25 kDa. It is purified 15621 times in comparison with initial nuclear extract.     

Partial purification of the nuclear estrogen receptor from chicken liver by affinity chromatography

The crude nuclear extract from the liver of estrogenized chickens contains 0.3–1 pmol/g tissue of the estrogen receptor. The receptor has been partially purified by ammonium sulphate precipitation and affinity chromatography on 17β-estradiol-17-hemisuccinyl-ovalbumin-Sepharose 4B. A 12% pure receptor preparation (2700-fold purification) with a yield of 17% could be obtained. The partially purified receptor has retained most properties which it displayed in cruder preparations, e.g. the dissociation constant of 10^?9?10^?10 M, the hormone specificity and the sedimentation coefficient of 3.9 S. The size (Stokes radius, 2.9 nm; molecular weight, 49 000) and the asymetry (f/f₀ = 1.10) of the receptor molecule, however, appear slightly reduced after the purification.     

First isolation of human UDP-D-xylose: proteoglycan core protein beta-D-xylosyltransferase secreted from cultured JAR choriocarcinoma cells

Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC, XT) initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein. In this study, we report the first isolation of the XT and present the first partial amino acid sequence of this enzyme. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcinoma cell culture supernatant. The isolation procedure included a combination of ammonium sulfate precipitation, heparin affinity chromatography, ion exchange chromatography, and protamine affinity chromatography. Among other proteins an unknown protein was detected by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman degradation. Searches with the amino acid sequences in protein and EST data bases showed no homology to known sequences. XT was enriched by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was specifically eluted with the antigen. In addition, XT was purified alternatively with an aprotinin affinity chromatography and was detected by Western blot analysis in the enzyme-containing fraction.     

Purification and some properties of polyphenol oxidase from the yam tubers, Dioscorea bulbifera

In a comparison of the polyphenol oxidase activity of various species of yam tubers the greatest enzyme activity was found in D. bulbifera. The enzyme was purified from acetone powder extracts of this plant. Ammonium sulphate fractionation, followed by ion exchange chromatography and gel filtration gave 22-fold purification. The final product gave a single band on polyacrylamide disc gel electrophoresis. The purified enzyme showed activity towards catechol, pyrogallol and dl-β-3,4-dihydroxyphenylalanine (dl-DOPA) and had a MW 115000 ± 2000. It was characterized by response to various inhibitors. β-Mercaptoethanol, dithioerythritol, l-cysteine, sodium metabisulphite and KCN inhibited strongly.     

Purification and characterization of a trypsin inhibitor from Senna tora active against midgut protease of podborer

Proteinaceous protease inhibitors have potential application in medicines, agriculture and biotechnology. Present study was undertaken to purify and characterize a proteinaceous protease inhibitor from a medicinal plant, Senna tora syn. Cassia tora. The inhibitor was purified by ammonium sulphate precipitation, anion exchange (Q-sepharose), affinity (trypsin-sepharose) and molecular exclusion (sephadex G-75) chromatography. Zymography and denaturing polyacrylamide gel electrophoresis revealed a single band of ∼20 kDa trypsin inhibitor. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix-assisted laser desorption ionization (MALDI) analyses revealed the presence of 19,725 Da (pI 4.60) and ∼19,900 Da (pI 4.57) isoform proteins in purified inhibitor. Protein identification by MALDI-peptide mass fingerprinting did not reveal high MASCOT (Matrix science) scores matching with previously known inhibitors. N-terminal amino acid sequence suggested this protein as a previously unreported inhibitor. Its dissociation constant (0.23 × 10⁻⁹ M) was indicative of a high affinity trypsin inhibitor. The inhibitor was stable over a broad range of pH (4–10) and temperature (30–60 °C). The purified inhibitor effectively inhibited total protease and trypsin-like activities of podborer (Helicoverpa armigera) midgut preparation. Hence, the inhibitor and its gene(s) can find application in combating against pest and protease dependent pathogens.     

Arylsulphatases in human brain: separation, purification, and certain properties of the two soluble arylsulphatases

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in the soluble subcellular fraction (105000g, 2 h) of human brain were partially purified by ammonium sulphate fractionation, ion exchange chromatography, and Sephadex gel filtration. Potassium-4-methylumbelliferone-sulphatase (MUS-sulphatase) adsorbed on DEAE-cellulose was purified approximately 700-fold over activity in the soluble fraction and the unadsorbed MUS-sulphatase was similarly purified approximately 600-fold. The arylsulphatase adsorbed to DEAE-cellulose exhibited a K_m value for MUS of 12.5 mM and a pH optimum of 5.7, whereas the unadsorbed arylsulphatase exhibited a K_m value for MUS of 8.3 mM and a pH optimum of 5.4. The molecular weights of the two enzymes were approximately 109,600 and 51,300, respectively. Sulphate (0.5 mM) showed pronounced mixed inhibition only of the unadsorbed arylsulphatase. Ag⁺ ions (0.25 mM) showed 96 per cent inhibition of the adsorbed arylsulphatase, whereas an activation of the unadsorbed arylsulphatase was observed.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Purification and properties of starch hydrolyzing enzymes in mature roots of sugar beets

Mature roots of sugar beets, which accumulate large amounts of sucrose but not starch, nevertheless contained acid and neutral amylases, judging from their pH optima, as well as pullulanase. Acid and neutral amylases were partially purified by procedures including fractionation with ammonium sulfate, ion exchange column chromatography, and gel filtration. Acid amylase was classified as an exoamylase, since it produced only glucose from soluble starch, amylopectin. β-limit dextrin, and rabbit liver glycogen. Neutral amylase was classified as an endoamylase, since it liberated maltose as the main product plus a small amount of glucose and oligosaccharides, and was capable of hydrolyzing β-limit dextrin. Pullulanase was purified to apparent homogeneity by procedures including fractionation with ammonium sulfate, Diethylaminoethyl-cellulose column chromatography and affinity chromatography. Pullulanase was capable of hydrolyzing soluble starch, amylopectin, β-limit-dextrin, and pullulan. Debranching of amylopectin was further evident by an increase in extinction coefficient, and by a shift of λ_max from 530 to 560 nm when the debranched amylopectin formed a complex with I₂-KI.