CM-Sephadex Column Chromatography Research Articles

Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313.

A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48 degrees C and 4.9, respectively. The enzyme was stable up to 60 degrees C and its pH stability was in the range of 5.0-8.0. The Km and Vmax of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, Ea, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a Ki of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a Ki of 9.5 mg/ml. The enzyme was inhibited by Hg2+, Mn2+, Fe3+ and Cu2+ and enhanced by Co2+ and Mg2+. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate.

Polyamine oxidase from leaves of Lilium longiflorum: Purification and properties

Summary Polyamine oxidase was purified to homogeneity from leaves of Lilium longiflorum using acetone powder, CM-Sephadex and hexamethylenediamine-Sepharose 4B column chromatography. The enzyme showed optimal pH of 5.5 and Km values of 16 and 6μmol · L−1 with spermidine and spermine, respectively. The enzyme was a monomer with molecular mass of 63,000 u (Da) as judged by sodium dodecyl sulfate Polyacrylamide gel electrophoresis. The enzyme appeared to contain 1 mol of flavin adenine dinucleotide per 63,000 g of the enzyme. Hexamethylenediamine was a potent competitive inhibitor with ki of 7 μmol · L−1. One mol per liter NaCl increased Vmax of the purified enzyme, similar to polyamine oxidase from oats, and also caused an increase of Km. Antibodies raised against the purified enzyme from Lilium longiflorum did not cross-react with polyamine oxidase from Avena sativa, Eichhonia crassipes, Gibasis pellucida, or Zea mays.

Application of thermostable xylanases from Humicola sp. for pulp improvement

Thermophilic Humicola sp. secreted thermostable xylanases when grown on wheat bran medium at 50°C. DEAE-Sephadex A-50 column chromatography of the crude xylanase separated three fractions of xylanase (I, II and III), xylanase I being homogeneous in polyacrylamide gel electrophoresis after CM-Sephadex column chromatography. The respective xylanases, including the crude xylanase, increased pulp brightness but xylanases II and III decreased the viscosity of the pulp due to CMCase activity. The crude xylanase contained lower CMCase activity than xylanases II and III.

Purification and characterization of alkaline serine protease from an alkalophilic Streptomyces sp.

SAP, an extracellular alkaline serine protease produced by Streptomyces sp. YSA-130, was purified to homogeneity by CM-Sephadex column chromatography and crystallization. The enzyme was a monomeric protein with a molecular weight of 19,000 as estimated by SDS-PAGE and gel filtration. The amino acid composition and amino-terminal sequence of SAP were similar to those of other bacterial serine proteases, i.e., Streptomyces griseus proteases A and B, Lysobacter enzymogenes alpha-lytic protease and Nocardiopsis dassonvillei subsp. prasina OPC-210 alkaline serine protease NDP-I. The optimum temperature and pH for the enzyme activity were 60 degrees C and 11.5. The enzyme was stable up 50 degrees C, and between pHs 4 and 12. The activity was inhibited by Ag+, Hg2+, Co2+, sodium dodecyl sulfate. N-bromosuccinimide, diisopropyl phosphorofluoridate (DFP), 2,3-butanedione, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), iodoacetate, N-ethylmaleimide (NEM), phenylmethanesulfonyl fluoride (PMSF), and phenylglyoxal.

Regulation of rat hepatic peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme by thyroid hormone

Rat hepatic t protein that is negatively regulated by thyroid hormone in nuclear globulin extract was characterized by the antibodies. The following evidence indicated that t protein is a peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (bifunctional enzyme). 1. Both proteins had an identical molecular size, and were immunologically indistinguishable from each other. 2. The t protein was abundant in mitochondrial fraction which contained abundant peroxisomes. 3. The amount of the t protein was increased by a peroxisomal proliferator. 4. The activity of the peroxisomal bifunctional enzyme corresponded to the t protein in CM-Sephadex column chromatography. The amount of peroxisomal bifunctional enzyme was increased by thyroidectomy and decreased by 3,5,3'- triiodo-L-thyronine treatment in the whole homogenate of rat liver. These results indicate that the levels of peroxisomal bifunctional enzyme were regulated by thyroid hormone in vivo.

Purification and properties of ascorbate oxidase from cucumber fruit

Abstract Ascorbate oxidase [AOD, EC 1.10.3.3], a copper-containing enzyme, was purified to homogeneity from cucumber fruit (Cucumis sativus) by CM-Sephadex and hydroxyapatite column chromatography and chromatofocusing. Isoelectric point (pI) of the purified enzyme was 8.3. Two other isoforms of AOD with alkaline pI values were also found. Purified AOD (MW 150,000) was a glycoprotein composed of two identical subunits (MW 70,000). N-terminal amino acid sequence was determined as Gly-Phe-Pro-Lys-Ile-Lys-His-Tyr-Lys-Trp-Asp-Val-Glu-Tyr-Met-Phe.

Two Apparent Molecular Forms of Bovine Lactoferrin

Bovine lactoferrin was prepared by CM-Sephadex column chromatography from defatted colostrum. When partially purified lactoferrin was analyzed by SDS-PAGE apparently two polypeptides of different size appeared. The polypeptides were transferred to a nitrocellulose sheet and visualized using antirabbit serum raised against the small polypeptide. Two polypeptides appeared clearly when stained by an immunological method. The color intensity of the two polypeptides was similar when the polypeptides were stained with Coomassie Brilliant Blue R-250. A high similarity of cyanogen bromide cleavage patterns between the two polypeptides was also observed in those stained with dye and immunologically visualized. Therefore, it is suggested that these polypeptides are lactoferrins. Comparison of the peptide patterns of the two lactoferrin molecules being deglycosylated suggested that sugar moieties may be one, but not all, of the causes of the size heterogeneity in lactoferrin. These results suggest that bovine colostrum contains two lactoferrin molecules of different size, although the physiological significance of the heterogeneity is not yet known.

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.

Isolation and characterization of three lethal and hemolytic toxins from the sea anemone Actinia equina L.

P. Maček and D. Lebez. Isolation and characterization of three lethal and hemolytic toxins from the sea anemone Actinia equina L. Toxicon 26, 441 – 451, 1988. — Lethal and hemolytic toxins were purified by acetone precipitation, Sephadex G-50, CM-cellulose and CM-Sephadex column chromatography from the tentacles and bodies of the sea anemone Actinia equina. The isolated toxins, with a mol. wt of 19,000 determined by SDS-polyacrylamide gel electrophoresis, differed only slightly in amino acid composition and had a high tryptophan content. The isoelectric points were estimated to be 9.8 for equinatoxin I and 10.5 for equinatoxins II and III. The pure toxins exhibited high lethal potency; the acute i.v. ld 50 in mice of equinatoxins I, II and III were 23, 35 and 83 μg/kg, respectively. The sigmoidal time course of hemolysis is characteristic of toxins.

An endodeoxyribonuclease activity in nuclei from rat-ascites hepatoma

The DNA in nuclei from rat-ascites hepatoma (AH) was rather resistant to endogenous endonucleolytic attack (autodigestion), compared with that in nuclei from normal rat liver (RL). In contrast, by micrococcal nuclease, the DNA in AH nuclei was cleaved in the same manner as in RL nuclei. A 0.6 M NaCl extract was prepared from RL or AH nuclei and subjected to Sephadex G-100 filtration. The resulting-nuclease fraction was separated further into two nuclease fractions, I and II, by CM-Sephadex column chromatography. The activity ratio of II to I was 7.1 for the RL and 2.0 for the AH nuclei. Moreover, the activity of fraction II from the AH nuclei was rather low, compared with that from the RL nuclei. Regenerating-liver nuclei from the normal rat were also assayed in the same way. The results obtained were very similar to those from the AH nuclei. In addition, each of fractions, I and II, cleaved pBR322 DNA of superhelical form; in other words, each had endonucleolytic ability.

Limited tryptic digestion of messenger RNA capping enzyme from Artemia salina. Isolation of domains for guanylyltransferase and RNA 5'-triphosphatase.

The partially purified preparation of messenger RNA guanylyltransferase from Artemia salina contains, as in the case of the rat liver enzyme (Yagi, Y., Mizumoto, K., and Kaziro, Y. (1983) EMBO J. 2, 611-615), the RNA 5'-triphosphatase activity which specifically removes the gamma-phosphoryl group from the 5'-triphosphoryl end of the newly synthesized mRNA molecule. The enzyme consists of a single polypeptide chain of Mr = 73,000 and forma a covalent enzyme-GMP complex as an intermediate for the guanylyltransferase reaction. Upon limited hydrolysis with trypsin, the enzyme-[32P]GMP complex is converted to a smaller 32P-containing fragment of Mr = 44,000. When the free enzyme, not complexed with GMP, is digested with trypsin under the same condition as above, the digests retain almost full activities of both guanylyltransferase and RNA 5'-triphosphatase and can form an enzyme-[32P]GMP complex of the size of Mr = 44,000 on incubation with [alpha-32P]GTP. Functional domains harboring the activities of guanylyltransferase and RNA 5'-triphosphatase are separated by gel filtration on a Sephacryl S-200 column at positions corresponding to Mr = 44,000 and 20,000, respectively. They can be separated completely from each other by CM-Sephadex column chromatography. While the native, undigested enzyme can transfer the GMP moiety to mRNA molecules with either triphosphoryl (pppN-) or diphosphoryl (ppN-)5'terminal, the purified Mr = 44,000 domain with the guanylyltransferase activity can utilize only the latter as an acceptor.

Molecular forms of glutathione S-transferase and UDP-glucuronyltransferase as hepatic preneoplastic marker enzymes.

Changes in molecular forms of glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDP-GT) as hepatic detoxicating enzymes were investigated during chemical hepatocarcinogenesis in the rat. The activity and the protein amount (formula; see text) itself of the GST-A form, which has fetal characteristics and is separable from other forms by CM-Sephadex column chromatography and by immunologic techniques, was much increased in gamma-glutamyl transpeptidase (gamma-GTP)-positive foci or hyperplastic nodules (HNs) induced by diethylnitrosamine and 2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene. The activity of enzyme 1 (late fetal form) of UDP-GT assayed with o-aminophenol (o-GT) also increased with increased number of the foci or HNs, while the activity of enzyme 2 (neonatal form) assayed with phenolphthalein (p-GT) changed but little. The foci and HNs were stained more strongly than the nonnodular areas immunohistochemically using the antibody against purified GST-A or o-GT. The two activities were also increased in well-differentiated hepatomas, but they were decreased in moderately and poorly differentiated hepatomas, and activating enzymes such as cytochrome P-450 were markedly decreased from HN. GST-A and o-GT differ from fetal enzymes such as those of carbohydrate metabolism in that they are inducible in the short-term by drugs, including carcinogens, and they show the highest activities in HNs, and so they may be considered as hepatic preneoplastic (PN) antigens.

Changes in activities of glutathione peroxidase and glutathione reductase during chemical hepatocarcinogenesis in the rat.

Changes in the activities of rat hepatic glutathione (GSH) peroxidase (GSH-Px) and GSH reductase (GR) were investigated during the induction of preneoplastic gamma-glutamyltransferase (gamma-GT)-positive foci and hyperplastic nodules by the administration of diethylnitrosamine followed by N-2-fluorenylacetamide. Activities of GSH-Px towards both cumene hydroperoxide (cumene-OOH) and hydrogen peroxide markedly decreased at an early stage of the above hepatocarcinogenesis, but the activity towards cumene-OOH again increased with the appearance of the foci and nodules, which were detectable as an increased gamma-GT activity. It was demonstrated by CM-Sephadex column chromatography and immunochemically that the increased activity towards cumene-OOH alone is due to the increased GSH-Px activity of GSH S-transferase B. GR activity and the total glutathione content in the liver also increased with increased preneoplastic foci and nodules. These results are discussed in connection with the protection mechanism(s) of these cell populations against damage by active oxygen and lipid peroxides produced in chemical hepatocarcinogenesis.

High-Performance Liquid Chromatographic Determination of Components of Bleomycin Preparations

A fast and sensitive method was developed for the quantitative determination of at least 10 components of pharmaceutical bleomycin sulfate preparations. The method is based on the reversed-phase high-performance liquid chromatographic (HPLC) separation of the components on a μBondapak C18 column with a mobile phase having a linear gradient of 10-40% methanol in aqueous 0.005 M 1-pentanesul-fonic acid at pH 4.3. With this assay, the average standard deviations for components A2 and B2 are 0.92 and 0.87, respectively, for a 7.5-22.5 × 110−3-mg sample. Regulatory agencies presently use the official Code of Federal Regulations (CFR), method, which is based on CM-Sephadex column chromatography. It was demonstrated that this CFR method does not separate the bleomycin A2 component from some other minor bleomycin components. After elution from the CM-Sephadex column, the “A2 component” was separated into five components by the HPLC method. Bleomycin A2 is stable under these HPLC conditions.

Isolation and properties of basic isoenzymes of horseradish peroxidase.

Six basic isoenzymes, E1 to E6, of horseradish peroxidase were isolated and purified by CM-Sephadex column chromatography, and were then crystallized. The crystalline basic isoenzymes had a lower molecular weights than the neutral isoenzyme C, but the molecular weights of their protein moieties were similar to that of the latter. The lower molecular weights of the basic isoenzymes were thus essentially due to smaller contents of carbohydrate. Of the six isoenzymes, four isoenzymes, E3 to E6, had extremely high pI values of over 12 and appreciably low contents of carbohydrate, 0.8 to 4.2%, whereas the isoenzymes E1 and E2 contained relatively large amounts of carbohydrates, 12.8 and 14.1%, respectively, and had lower pI values, 10.6. The amino acid compositions of the basic isoenzymes were different from one another, and the differences suggested that the isoenzymes were allelic. The relatedness of the amino acid compositions of these isoenzymes is discussed and the compositions are compared with those of other peroxidases. Finally, one of the six isoenzymes, E4, was obtained as large single crystals suitable for X-ray structural analysis, and the preliminary crystallographic data are presented.

Purification and subunit structure of glutathione reductase from human leukocytes

Glutathione reductase from human leukocytes has been purified 570-fold by using the methods of ammonium sulfate fractionation (35–70%), CM-Sephadex column chromatography, and affinity chromatography. The molecular weight of the enzyme was determined by gel filtration on Sephadex G-200 and found to be 120,000 ⊣ 5000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme had a molecular weight of 19,000 ± 2000 when dissolved in 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. Velocity versus pH curve indicated that the enzyme may be a diprotic system.

Factor-dependent dissociation of wheat germ ribosomes

Ribosome dissociation factor has been found in wheat germ acetone powder extracts. Further purification of the crude extract by pH and ammonium sulfate fractionations, DEAE-cellulose and CM-Sephadex column chromatography has resulted in the separation of two active fractions. The possibility that ribosome dissociation activity exhibited by either fraction is due to a protease or nuclease is considered unlikely, based on results of experiments involving ribosome dissociation kinetics, subunit structural integrity, and treatment with a serine protease inhibitor. Wheat germ ribosome dissociation factor is not species-specific. Dissociation factor from both fractions will promote the dissociation of Escherichia coli 70-S as well as Artemia salina 80-S ribosomes. Although both dissociation factor activities show the same dependence on K + and Mg 2+ for optimal activity, the two activities exhibit significant differences in their sensitivity to sulfhydryl reagents and heat, and in their dependence on incubation temperature for activity. Certain properties of both factors suggest that neither factor is initiation factor eIF-3; however, the possibility that one or both factors are subunits of initiation factor eIF-3 remains to be determined.

Cartilage-derived factor (CDF): I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of 35SO 4 −2, [ 3H]-glucosamine and [ 3H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.

Single-strand-specific nuclease from the nucleoplasm of rye germ nuclei.

The localization of DNAase activity in rye germ nuclei revealed 48% of the total nuclear activity in the nucleoplasm and 4% in the chromatin.2. The endonuclease present in the nucleoplasm was purified more than 300‐fold as compared with the specific activity in homogenized nuclei by means of ammonium sulphate fractionation and CM‐cellulose and CM‐Sephadex column chromatography. The preparation gave a single band on polyacrylamide gels. Its molecular weight is 45000, pH optimum 5.4. The preparation does not exhibit activity of non‐specific phosphomonoesterase and phosphodiesterase.3. The nuclease catalyses the hydrolysis of denatured DNA and RNA to acid‐soluble 5′‐phoshate‐terminated products and cleaves the phosphomonoester linkage of 3′AMP.4. Synthetic polyribonucleotides are hydrolysed at a rate decreasing in the order: poly(U) > Poly(A) > Poly(C) > Poly(G).5. The endonuclease shows a high preference for single‐stranded nucleic acids. It does not depolymerise double‐stranded poly(U)· poly(A).6. The nuclease attacks covalently closed circular bacteriophage PM2 DNA, plasmid ColE1 and plasmid pBR322 DNA, converting it initially into the open‐circular (relaxed) form and subsequently into the linear form. This reaction is strongly dependent on ionic strength.7. The enzyme nicks the supercoiled DNA molecule of phage PM2 in only one place. At high enzyme concentrations several nicks in such a molecule are observed.

Crystallization and properties of cathepsin B from rat liver.

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugal analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22,500 (+/- 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with alpha-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.