Purification and properties of cytoplasmic and mitochondrial hexokinase from the rabbit brain

The soluble Hexokinase from rabbit brain was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The properties of the purified cytoplasmic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in M.W., pI and electrophoretic mobility. However, the temperature dependence of activity and specificity for several hexose substrates were markedly different.     

Electrophoretic data in designing strategies for purification and identification of a highly polymorphic bacterial esterase

We have purified a bacterial enzyme, designated esterase M, by tailoring an efficient and rapid strategy with information derived from titration curves of proteins in crude extract. The pH-dependent stability of the enzyme activity observed by titration pattern allowed an acidic pH treatment of extract and a cationic exchange chromatography at pH 4.1. These two steps were followed by an anionic exchange chromatography and a preparative electrophoresis. Thus, the enzyme was purified about 2000-fold within two days with a recovery of 13.3%. The electrophoretic variants of esterase M were investigated for their molecular relationship through the specific effect of antibodies on esterase electrophoretic pattern (immunosubtractive electrophoresis) which is applicable to large series of samples. By this process, we have demonstrated the presence of common antigenic determinants among the electromorphs of esterase M produced by the three species of motile Aeromonas.     

短小芽孢杆菌2080碱性蛋白酶的纯化与性质

短小芽孢杆菌（Bacillus pumilus）2080碱性蛋白酶的发酵液经超滤、硫酸铵沉淀、CM Sepharose Fast Flow和DEAE Sepharose Fast Flow离子交换层析得到了纯化的组分。SDS-PAGE电泳分析显示其分子量约为61kDa。酶学性质研究表明,该纯化酶的最适pH为10．5,最适温度为50℃。     

Comparison of the properties of purified mitochondrial and cytosolic rat kidney transamidinase

1. Mitochondrial rat kidney transamidinase was solubilized by two extractions with the surfactant Zwittergent 3-14. 2. Mitochondrial and cytosolic forms of rat kidney transamidinase were purified by chromatography on DEAE-Trisacryl M, phenyl-Sepharose Cl-4B and hydroxylapatite columns. 3. The specific activity of purified mitochondrial enzyme was significantly higher than purified cytosolic enzyme. 4. The subunit molecular mass, the electrophoretic mobility under nondenaturing conditions, and the activation energy were similar for purified mitochondrial and cytosolic transamidinase.     

Separation of two forms of glutamate synthase in leaves of tomato (Lycopersicon esculentum)

Two forms of glutamate synthase, one dependent on NAD(P)H, and the other on ferredoxin, have been completely separated by ionic exchange chromatography on DEAE cellulose. The NAD(P)H dependent enzyme was further purified by affinity chromatography with Blue Sepharose, showing Km values of 0.5 mM, 0.3 mM and 1.7 μM for glutamine, 2-oxoglutarate and NADH, respectively. Ferredoxin dependent enzyme was also purified to electrophoretic homogeneity; the Km values were 0.5 mM, 0.2 mM and 0.2 μM for glutamine, 2-oxoglutarate and ferredoxin, respectively. These results support the glutamine synthetase-glutamate synthase pathway for nitrogen assimilation.     

Purification and characterization of human prostatic acid phosphatase.

Human prostatic acid phosphatase (orthophosphoric monoester phosphohydrase, EC 3.1.3.2) is purified to homogeneity by standard procedures which include CM-Sephadex, Con A affinity chromatography and gel filtration. The purified enzyme is antigenically specific and has a M.W. of 100,000 with subunit M.W. of 48,000. However, the enzyme exhibited charge heterogeneity. Two major electrophoretic or chromatographic isozymic forms of PAP were separated by DEAE-Sephadex chromatography and their immunochemical identity was studied by immunodiffusion before and after the neuraminidase digestion. Quantitative precipitin and inhibition experiments showed immunological identity of the two chromatographic isozymes. Immunologic specificity of this enzyme resides on the protein moiety rather than the carbohydrate residue, although the latter group is mostly responsible for the charge group heterogeneity of the enzyme.     

Studies on Amylases of Aspergillus oryzae Cultured on Rice

α-Amylase was purified from a culture of Aspergillus oryzae on steamed rice by means of ion exchange chromatography on DEAE-Sephadex, and the purified enzyme was crystallized with ammonium sulfate. The preparation was found to be homogeneous by means of sedimentation and disc electrophoretic analyses. The enzyme was revealed to have strong α-amylase activity by the dinitrosalicylate method and the iodine color method. Large single crystals of the enzyme were prepared by making the concentrated enzyme solution to 0.41 saturation of ammonium sulfate at pH 5.0. A brief communication on the preliminary X-ray crystallography was also presented.     

Multiple forms of glutamine synthetase in Bacillus brevis AG 4

Glutamine synthetase in Bacillus brevis AG 4, a Gram-positive spore forming bacteria, has been found to exist in multiple molecular forms. It was purified to electrophoretic homogeneity by single-step Blue Sepharose affinity chromatography. The native enzyme has a molecular weight of 600,000 with subunits of 50,000. The enzyme samples purified from different stages of growth differed in Mg2+ sensitivity and other kinetic properties. Four different enzyme samples selected on the basis of Mg2+ sensitivity showed distinct mobilities at pH 6.3 on PAGE using discontinuous buffer system. A correlation amongst Mg2+ sensitivity, electrophoretic mobility, and kinetic properties was highly suggestive of multiple forms of glutamine synthetase in Bacillus brevis arising due to modification.     

Human adenosine deaminase. Purification and subunit structure.

Human erythrocyte adenosine deaminase has been purified approximately 800,000-fold to apparent homogeneity using antibody affinity chromatography. The enzyme was shown to be a single polypeptide chain with an estimated molecular weight of approximately 38,000. The three electrophoretic forms of erythrocyte adenosine deaminase purified simultaneously by this technique were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Several properties of the highly purified adenosine deaminase including pH optimum, Km for substrate, Ki for product, Stokes radius, sedimentation coefficient, and apparent substrate specificity were identical with the properties observed with an impure preparation of the enzyme.     

The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes.

Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.     

Isolation and Characterization of S-Adenosyl-L-Methionine:Tetrahydroberberine-cis-N-Methyltransferase from Suspension Cultures of Sanguinaria canadensis L

As part of a continuing study of the induction of alkaloid biosynthesis, we report the isolation to homogeneity and characterization of S-adenosyl-L-methionine:tetrahydroberberine-cis-N-mehtyltransferase from suspension cultures of Sanguinaria canadensis that were induced to produce alkaloids by hormone depletion. This enzyme catalyzes the stereospecific transfer of a methyl group from S-adenosyl-L-methionine to the tertiary nitrogen of the protoberberine alkaloid tetrahydroberberine (canadine). The enzyme was purified 315-fold by ammonium sulfate precipitation, gel permeation chromatography, affinity dye chromatography, and both diethylaminoethyl and Mono-Q ion-exchange chromatography. The enzyme was further purified to an optimum specific activity of 225 nkat/mg of protein (3500-fold) and electrophoretic homogeneity by native polyacrylamide gel electrophoresis (PAGE). In contrast to previous reports with partially purified enzyme, the isolated protein was found to have a pH optimum of 7.0, a temperature optimum of 25 to 30[deg]C, and an isoelectric point of 5.1. Furthermore, the molecular weight of the homogeneous protein was found to be 39,000 by sodium dodecyl sulfate-PAGE. The homogeneous enzyme preferred tetrahydroberberine over all other substrates tested, showing an apparent Km of 2.1 [mu]M, but also showed partial activity with tetrahydrojatrorrhizine and tetrahydropalmatrubine.     

Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography.

1. Affinity elution chromatography was used to purify phosphoglycerate kinase from a variety of sources. The choice of buffer pH for the chromatography was made according to the relative electrophoretic mobility of the enzyme from the species concerned. 2. Outlines of the methods used to isolate the enzyme from over 20 sources are presented. The enzyme was purified from the muscle tissue of a variety of mammals, fish and birds, from liver of several animals, from yeast, Escherichia coli, and plant leaves. The more acidic varieties of the enzymes were purified by conventional gradient elution from ion-exchangers as affinity elution procedures were not applicable. 3. The structural and kinetic parameters investigated show that phosphoglycerate kinase is evolutionarily a highly conservative enzyme; there were few differences in properties regardless of source or function (glycolytic, gluconeogenic or photosynthetic). 4. A detailed comparison of the enzyme preparations purified from bovine muscle and bovine liver failed to detect any significant differences between them; the evidence indicates that they are genetically identical.     

Characterization of Polyphenol Oxidase from the Latex of Opium Poppy

Polyphenol oxidase from the latex of opium poppy was purified to the electrophoretic homogeneity by affinity chromatography using p-aminobenzoic acid as a ligand coupled to Sepharose CL-4B by divinyl sulphone activation method. The purified enzyme was used to prepare the polyclonal antibodies. The purified latex PPO exhibited high diphenolase activity in comparison with almost unmeassurable monophenolase activity. Both of these activities were sensitive to the activation with sodium dodecyl sulphate. Two isoforms (65 and 40 kDa) of latex PPO were separated by the gel filtration. There were no differences in substrate specifity (weak monophenolase and high diphenolase activity) and sensitivity to inhibitors between these isoforms, but they showed differences in electrophoretic mobility. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the branching enzyme

A branching enzyme was extracted from the mycelia of Neurospora crassa and was purified to electrophoretic homogeneity by procedures including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose 4B column chromatography and gel filtration on Toyopearl HW-55S. The final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. The molecular weight of this enzyme was estimated to be 80,000 by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. The amino acid composition and the carbohydrate content of this enzyme were analyzed. The isoelectric point of this enzyme determined by polyacrylamide gel isoelectrofocusing was 5.6. The branching activity of the enzyme was confirmed by its action on amylopectin as well as by the combined action of this enzyme and N. crassa glycogen synthase. The action of this enzyme on amylopectin decreased the wavelength of the absorption maximum of the glucan-iodine complex, and increased the amount of the short unit chains of the debranched product. The product obtained by the combined action yielded beta-limit dextrin upon hydrolysis with beta-amylase. No multiplicity was found for the branching activity either by chromatography or by electrophoresis.     

Purification of swine kidney alkaline phosphatase by immunoaffinity chromatography

A homogeneous alkaline phosphatase preparation was obtained from swine kidney cortex by a simple purification step of immunoaffinity chromatography. The enzyme was purified 426 times that of the initial acetone powder with a recovery of 69.6% and a specific activity of 1206 units/mg of protein. The sodium dodecyl sulfate-gel electrophoretic pattern showed a single 80,000-M_r protein band as the monomer of the purified enzyme.     

Simultaneous purification by affinity chromatography of rat liver mitochondrial aspartate aminotransferase and malate dehydrogenase and electrophoretic properties

Mitochondrial aspartate aminotransferase and malate dehydrogenase were purified to homogeneity from rat liver by the use of aspartate-coupled Sepharose, ion exchange, and Blue Sepharose chromatography. This procedure permits rapid preparation of these enzymes. The pI of each enzyme was determined and anomalous electrophoretic properties of aspartate aminotransferase were described.     

Purification and biochemical properties of glutathione S-transferase from Lactuca sativa

A glutathione S-transferase (GST) from Lactuca sativa was purified to electrophoretic homogeneity approximately 403-fold with a 9.6% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the enzyme was significantly inhibited by ShexylGSH and S-(2,4-dinitrophenyl) glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards ethacrynic acid. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide.     

PURIFICATION AND PROPERTIES OF BRAIN ALKALINE PHOSPHATASE

Abstract— Alkaline phosphatase from sheep brain has been purified to homogeneity. The method includes butanol extraction, fractional ethanol precipitation, ion-exchange chromatography on DEAE-cellulose, and on DEAE-Sephadex followed by Sephadex G-200 filtration. By these steps, the enzyme is purified 22,920-fold with 15% recovery. The homogeneous enzyme is shown to be a sialoglycoprotein in nature. Neuraminidase treatment reduces the electrophoretic mobility of the enzyme. The enzyme shows pyridoxal phosphate phosphatase activity along with p -nitrophenylphosphate phosphatase activity. Both these compounds behave as mutual alternate competitive substrates. The general properties of the enzyme are described.     

Large-scale purification of fungal glucoamylases using anion-exchange resin chromatography

Anion-exchange chromatography on polystyrene resin is shown to be more effective than DEAE-cellulose for purification of glucoamylase from crude enzyme extracts of Aspergillus awamori or from commercial preparations. The glucoamylase from A. awamori culture medium was purified to electrophoretic homogeneity with yields approaching 80%.     

Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37

A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with alpha-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50 degrees C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-L-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.