CM-Sepharose CL-6B Research Articles

NAD +-glycohydrolase from Streptococcus pyogenes shows cyclic ADP-ribose forming activity

NAD +-glycohydrolase from Streptococcus pyogenes was purified by successive chromatography on CM Sepharose CL-6B, Sephacryl S-200 HR and hydroxyapatite. The purified enzyme possessed synthesis and hydrolysis activities of cyclic ADP-ribose (cADPR), a newly found second messenger for Ca 2+ mobilisation, along with cleavage activity of the ribose-nicotinamide bond in NAD +.

NAD(+)-glycohydrolase from Streptococcus pyogenes shows cyclic ADP-ribose forming activity.

NAD(+)-glycohydrolase from Streptococcus pyogenes was purified by successive chromatography on CM Sepharose CL-6B, Sephacryl S-200 HR and hydroxyapatite. The purified enzyme possessed synthesis and hydrolysis activities of cyclic ADP-ribose (cADPR), a newly found second messenger for Ca2+ mobilisation, along with cleavage activity of the ribose-nicotinamide bond in NAD+.

Purification and characterization of piscivorase i and ii, the fibrinolytic enzymes from eastern cottonmouth moccasin venom ( Agkistrodon piscivorus piscivorus)

Fibrinolytic enzymes, piscivorase I and II, were isolated from Agkistrodon piscivorus piscivorus (eastern cottonmouth moccasin) venom using gel filtration on Bio-Gel P-100 and ion-exchange chromatography on CM-Sepharose CL-6B. The mol. wts of these proteases, piscivorase I and II, are 23,400 and 29,000 and isoelectric points are 6.6 and 8.5, respectively. These fibrinolytic enzymes were homogeneous by SDS-polyacrylamide gel electrophoresis. Piscivorase I readily cleaved the Aα- and Bβ-chain of fibrinogen, but piscivorase II cleaved readily the Aα-chain and more slowly the Bβ-chain. These fibrinolytic enzymes were activated by Ca 2+, Mg 2+ and Ba 2+, but inhibited by Zn 2+, Cu 2+ and Mn 2+. Both fibrinolytic enzymes were also inhibited by cysteine, β-mercaptoethanol, and by metal chelators such as EDTA and EGTA, but not by benzamidine, phenylmethanesulfonyl fluoride (PMSF), soybean trypsin inhibitor and aprotinin. These fibrinolytic enzymes did not act like thrombin, plasmin and kallikrein, using specific chromogenic substrates. Neither fibrinolytic enzyme induced platelet aggregation, and piscivorase I showed low haemorrhagic activity at dosages of 55 μg.

Purification of peroxisomal acyl-CoA: dihydroxyacetonephosphate acyltransferase from human placenta

The peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT) was extracted from human placental membranes using CHAPS as a detergent in the presence of 1 M KCl. Prior to assay dipalmitoylphosphatidylcholine was added to the sample as eluted from the various columns in order to stabilize the protein for subsequent enzyme activity measurements at 37°C. The enzyme was purified from the placental membrane using octyl-Sepharose CL-4B chromatography, Hydroxyapatite HTP chromatography, CM-Sepharose CL-6B, PBE 94 chromatofocusing and TSK G3000 SW size exclusion chromatography. A final purification of more than 8000-fold with respect to the placental membranes was achieved with a final yield of about 5%. Upon chromatofocusing the peak of activity eluted at a pH of 5.1–5.3 indicating a low isoelectric point. A native M r of 60–80 kDa was calculated from HPLC size exclusion chromatography. SDS-PAGE of the final purified fraction showed one major band with a M r of 65 kDa. These results suggest that DHAPAT is a monomeric protein. A polyclonal antiserum raised against the purified fraction was prepared in rabbits. Immunoprecipitation experiments showed complete precipitation of DHAPAT activity in fractions prepared from human placenta, liver and skin fibroblasts. Immunoprecipitation was also used to determine the residual amount of DHAPAT protein in liver from a patient with the Zellweger syndrome. A value of about 10% was found, which closely corresponds to the residual amount of enzyme activity.

Isolation and characterization of a serine proteinase, inactivating m-subunit of lactate dehydrogenase, from Penicillium citrinum KE-1.

A selective inactivating enzyme for the m-subunit of lactate dehydrogenase (LDH) was found in the culture filtrate of Penicillium citrinum KE-1, newly isolated from soil. The enzyme was purified from the culture filtrate by ammonium sulfate fractionation, column chromatography on CM-Sepharose CL-6B, and gel filtration on Sephadex G-100. The purification was 124-fold with an activity yield of 81%. The purified enzyme gave a single band, corresponding to a molecular weight of 32,000, on SDS polyacrylamide gel electrophoresis, and the isoelectric point was 9.5. The enzyme specifically inactivated the m-subunit of LDH but showed no activity on the h-subunit of LDH. The enzyme, named KE-1 proteinase, proved to be a serine-type proteinase. Limited proteolysis of native m-subunit of LDH was assumed to result in a loss of enzyme activity.

Isolation and Characterization of Pectin Methylesterase from Papaya

Pectin methylesterase (PME) (EC 3.1.1.11) has been purified to apparent homogeneity from ripe papaya fruits. The purification protocol consisted of ammonium sulphate precipitation (60-80%) and cation exchange chromatography in CM Sepharose CL-6B and Mono S. Papaya PME consists of two components (PME 1 and PME 2), which have been shown to be isoenzymes by Ferguson plot analysis. The molecular weight of the enzyme is 27,000 while its isoelectric point is greater than pH 9.0. The N-terminal sequences of PME 1 and PME 2 are SVVTPNAVVADDGVFXFKTG. Both PME 1 and PME 2 showed optimum activities at pH 8.0 and 35°C. The average K m s of PME 1 and PME 2 are 0.0071 and 0.0166 g/liter pectin respectively, while the corresponding average V maxs are 741 and 800 μmol methanol/min/mg protein, respectively. Papaya pectin methylesterase is activated by cations, but the effect is more pronounced for divalent than monovalent cations. Inhibition studies showed that sucrose is a noncompetitive inhibitor while p-chloromercuribenzoic acid has no significant effect on its activity.

Protease D from the sarcocarp of honeydew melon fruit

A serine protease was purified from commercially available honeydew melon fruit ( Cucumis melo L. var. inodorus Naud) by five steps including CM-Sepharose CL-6B column chromatography. At the first CM-Sepharose CL-6B chromatography four active fractions appeared. No difference in enzymatic properties and M r in each fraction was found. Finally, honeydew melon protease D was isolated from the major active fraction D. A M r of 50 000 was determined for protease D on SDS-PAGE and gel filtration. Protease D was a glycoprotein. The protease exhibited a remarkable stability, even in 8 M urea or 2 M guanidine hydrochloride for 24 hr. The optimum pH of the enzyme was 11 at 35° using casein as substrate. The enzyme was strongly inhibited by diisopropyl fluorophosphate and was not inhibited by metal-chelating reagents or cysteine protease inhibitors. The enzymatic properties of honeydew melon protease D were similar to those of cucumisin [EC 3.4.21.25], except for its stability at high temperature, wide pH range and against detergents.

Isolation and amino acid sequence of a chemotactic protein, LECT/interleukin 8, from a human myeloid leukemia cell line, ML-1

We looked for chemotaxin/interleukin 8 (CT/IL-8) activity in the culture fluids of 97 human leukemia cell lines and found it in two of the T cell lines, six of the myeloid cell lines, and one of the normal B-cell lines. It was particularly strong in the culture fluids of two cell lines. These cell lines secreted a chemotactic protein into the culture fluids under certain conditions of stimulation with phorbol-12-myristate 13-acetate (PMA), lipopolysaccharide, or hemagglutinin-P. A myeloid leukemia cell line, ML-1, secreted an inducible chemotaxin when stimulated with PMA (1 ng/ml) for 24 h. We purified the chemotaxin from ML-1 cell culture fluid using an improved procedure: concentration with DEAE-Sepharose CL-6B and CM-Sepharose CL-6B, CM-Sepharose column chromatography, and reverse-phase 5TMS-300 column on HPLC with the retention time coinciding with that of LUCT/IL-8 [Suzuki et al. 1989, J. Exp. Med. 169, 1895]. The yield was 200 μg protein from 6 liters of the culture fluid. The N terminus of CT/IL-8 was AVLPR-SAKELRXQXIKTYSK —, the same as that of LUCT/IL-8, which is constitutively secreted from lung giant cell carcinoma LU65C cells. The optimal concentration in the chemotactic activity of CT/IL-8, equivalent to that of bacterial chemotactic peptide fMet-Leu-Phe (10 nM), was found to be 5 nM. The results show that this chemotaxin is identical to LUCT/IL-8.

Evaluation of cation exchange chromatography for the isolation of M glycoprotein from histoplasmin

Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.

Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei

During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 μg polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues.

Purification and characterization of two extracellular β-glucosidases from Trichoderma reesei

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.

A two-substrate kinetic study of peroxidase cationic isoenzymes in barley malt

Ten active cationic peroxidase isoenzymes were identified by polyacrylamide gel electrophoresis of extracts from malt of Triumph barley. These were partially separated on CM-Sepharose CL-6B and their properties examined. The five active peaks had different pH optima for the oxidation of 2,2′-azinobis(3-ethylbenzthiazoline 6-sulphonate) (ABTS), ranging from pH 3.25 to 3.73, and different inactivation rates at 55°, two being significantly more stable than the rest. A two-substrate kinetic study revealed a parallel pattern of double reciprocal plots for some of the active peaks, a converging pattern for others and a range of true K m values for the isoenzyme peaks varying from 76 to 710, μM for hydrogen peroxide and 2–310, μM for ABTS. The significance of the observations is discussed in relation to the known activity of peroxidase during brewery mashing.

Continuous separation and concentration of proteins using an annular chromatograph.

A continuous rotating annular chromatograph with rotating feed nozzles and product collectors was used to separate two kinds of proteins, (myoglobin and hemoglobin). Simultaneously, both proteins could be concentrated by using two kinds of eluents. The cation exchange resin, CM-Sepharose CL-6B was used as an adsorbent. The adsorption forces of both proteins on CM-Sepharose CL-6B were dependent on the value of pH in the buffer solution. Therefore, both proteins could be separated and concentrated by changing pH in the eluent. Experimental results could be predicted by numerical solutions.

A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.

Purification and Characterization of Oyster Glucose 6-Phosphate Dehydrogenase.

Glucose 6-phosphate dehydrogenase, (EC 1. 1. 1. 49; Glc-6-P DH) in oyster Crassostrea gigas was extracted with 10mM Tris-HCl buffer solution and purified by ammonium sulfate fractiona-tion, Sepharose 6B gel filtration, and DEAE-Sephadex A-50, and CM-Sepharose CL-6B chromato-graphy. A single fraction exhibiting Glc-6-P DH activity was obtained by CM-Sepharose CL-6B chromatography. The purified Glc-6-P DH was electrophoretically homogenous. The optimal pH for hydrolysis of Glc-6-P was 8. The temperature which inactivated 50% of enzyme was 47°Cin 15-min treatment. The molecular weight was 204, 000. Beyond 35°C the activation energy of Glc-6-P DH was 5, 400 cal/mol, and below 35°C it was 10, 900 cal/mol. The enzyme was inhibited by Zn2+ and Hg2+ ions in low concentration and by Co2+ and Cu2+ ions in high concentration. How-ever, it can be activated by adding Ca2+, Mg2+, and Mn2+ ions in high concentration.

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake ( Lachesis muta muta)

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose Cl-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A 2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22 300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectivity the A α-chain of fibrinogen, leaving the Bβ- and γ-chains unaffected. LHF-II is activated by Ca 2+ and inhibited by Zn 2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.

Isolation of a ribosome‐inactivating and abortifacient protein from seeds of Luff a acutangula

A glycoprotein with a molecular weight of 28,000 as estimated by SDS-polyacrylamide gel electrophoresis was isolated from seeds of Luffa acutangula using a procedure that involved acetone precipitation, ion exchange chromatography on CM Sepharose CL-6B and gel filtration on Sephadex G-50. In immunodiffusion studies it was found to be immunologically distinct from abortifacient proteins isolated from other members of the Cucurbitaceae family including Momordica charantia, Momordica cochinchinensis, Trichosanthes kirilowii and Trichosanthes cucumeroides. There were some differences in amino acid composition among the proteins although there was a gross similarity. The protein from L. acutangula was capable of inducing mid-term abortion in mice and inhibiting protein synthesis in a cell-free system.

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM OYSTER (CRASSOTREA GIGAS)

Proteases in oyster (Crassotrea gigas) were extracted with 10 mM Tris-HCl buffer solution and purified by ammonium sulfate fractionation, Sephadex G-150 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Three fractions with caseinolytic activity, named I, II and III, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The three proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that protease I was a carboxypeptidase A-like enzyme; II and III were trypsin-like enzymes. The optimal pH of protease I for hydrolysis of hippuryl-L-phenylalanine was 9.0, II and III for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester (TAME) was 8.0. The temperatures which inactivated 50% of enzymes were 78°C for protease I in 30 min; 50 and 52°C for protease II and III, respectively, in 5 min. The molecular weights of proteases I, II and III were 23,000, 34, 400 and 31, 000, respectively.

A Specific Glutathione S-Transferase Isozyme Occurring in Hereditary Hyperbilirubinuria Rats

A glutathione S-transferase isozyme which is absent in normal rat liver has been isolated from the hereditary hyperbilirubinuria rat liver cytosol. The enzyme was purified to apparent homogeneity by GSH-affinity chromatography and HPLC on CM-Sepharose CL-6B. It is a heterodimer of two non-identical subunits, i.e., subunit 2 and a previously uncharacterized subunit referred to here as subunit Yx. Immunoblot analysis indicated that GST 2-Yx belongs to the alpha class. GST 2-Yx is characterized by its 4-fold higher activity towards 4-hydroxy-non-2-enal, compared to that of GST 2-2.

Purification and Some Properties of a Thermostable Metal Proteinase Produced by Thermomicrobium sp. KN-22 Strain.

An extreme thermophile that produces a heat-stable proteinase was isolated from hot-spring water and classified as Thermomicrobium sp. KN-22 (growth temperature, 50-83 degrees C; and optimum growth temperature, 70 degrees C). The proteinase was purified from the culture broth of this strain by fractionation with ammonium sulfate, chromatography on columns of DEAE-cellulose and CM-Sepharose CL-6B, and HPLC on TSKgel CM-5PW. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and a single peak after HPLC (yield 8.8%). The enzyme had maximum activity at pH 8.5 and at 75 degrees C and it was stable up to 60 degrees C. The molecular weight of the enzyme was 35,000 by SDS-PAGE. Since the enzymatic activity was completely inhibited by EDTA, o-phenanthroline, and phosphoramidon, it appears that the enzyme is a metal proteinase.