Sepharose 4B Column Research Articles

Tissue fibronectin is an endogenous ligand for galectin-1.

A 14K beta-galactoside-binding lectin (galectin-1) is present in many animal tissues. In a search for endogenous ligands, we surveyed galectin-1-binding proteins in human placenta. Extract of human placenta with 2 M urea was applied to a Sepharose 4B column conjugated with galectin-1 purified from frog (Rana catesbeiana) eggs. Two major proteins eluted with 100 mM lactose from the column-bound fraction showed apparent molecular masses of 220 and 180 kDa on SDS-PAGE under reducing conditions. Western blotting analysis using monoclonal antibodies indicated that these proteins were fibronectin and laminin, respectively. Most placental and amniotic fibronectins bound strongly to the column, whereas almost all plasma fibronectin passed through the column. The galectin-1, fibronectin and laminin were immunohistochemically shown to be co-localized in the extracellular matrix of placental tissue. In a cell attachment assay, rhabdosarcoma cells adhered to a plate coated with placental fibronectin, even in the presence of GRGDS peptide, if galectin-1 were also present. This adhesive effect of galectin-1 was inhibited by lactose. These results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectin-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular matrix interaction.

Purification of a Phenobarbital-Inducible Morphine UDP-Glucuronosyltransferase Isoform, Absent from Gunn Rat Liver

A morphine UDP-glucuronyltransferase (morphine UGT PB) was purified from liver microsomes of Sprague-Dawley rats treated with phenobarbital. UDP-glucuronyl-transferases in the liver microsomes were solubilized with Emulgen 911 and separated by ω-(β-carboxypropionylamino)octyl Sepharose 4B column chromatography, which has been developed in our laboratory. Morphine UDP-glucuronyltransferases were eluted into two fractions, Peak I and Peak II, which have different substrate specificities. Morphine UGT PB was purified by two times of Chromatofocusing from Peak II which was more specific to morphine. The purified morphine UGT PB gave an apparent p I of 8.0 on chromatofocusing and displayed a subunit molecular weight of 55 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the glucuronidation of 3-hydroxyl group of morphine and small extent of 4-hydroxybiphenyl, but not of androsterone, bilirubin, chloramphenicol, codeine, 4-methylumbelliferone, 4-nitrophenol, testosterone, and 6-hydroxyl group of morphine. The N-terminal amino acid sequences of morphine UGT PB were identical to those of UGT1*01P which is deficient to homozygous Gunn rat. Peak II was absent from the fraction of ω-(β-carboxypropionylamino)octyl Sepharose 4B column chromatography of liver microsomes of Gunn rats treated with phenobarbital, whereas morphine UGT in Peak I was PB-inducible in Gunn rats. Present results suggest that an isoform of morphine UDP-glucuronyltransferase belongs to the UGT1 family and is phenobarbital-inducible.

Fast Purification ofPhaseolus VulgarisIsolectins

Phytohemagglutinin from red kidney bean has been purified by affinity chromatography on a human alpha 1-acid glycoprotein Sepharose 4B column. Further purification of the hemagglutinin's five isolectins was achieved on a Mono S column with an 86% protein recovery. Each sequentially eluted isolectin from the ion exchange column displayed either hemagglutinating or mitogenic activity. The main activity of each fraction was the result of the combination of varying proportions of the L and E subunits.

Purification and Partial Characterization of a Heparin-Binding Lectin from the Marine Clam Anadara granosa

The heparin-binding lectin, Anadarin MS, from the plasma of the marine clam Anadara granosa is purified through affinity chromatography on heparin-Sepharose 4B followed by gel filtration on a Sepharose 6B column. The purified lectin is a pentameric protein of native Mr 300 kDa and is composed of identical subunits of 60 kDa. The pI value of this Ca2+-dependent lectin is 6.2. Anadarin MS agglutinates normal rabbit erythrocytes but not that of human. Aspartic acid, glutamic acid, histidine and glycine are the predominant amino acids. Unlike other reported heparin-binding lectins, Anadarin MS exhibits a unique and strict specificity for iduronic acid containing glycosaminoglycans. This lectin agglutinates infective promastigotes of Leishmania donovani exclusively and can therefore be used as a novel biochemical surface marker for this parasite.

Cock seminal plasma acid phosphatase: Active site directed inactivation, crystallization and in vitro denaturation-renaturation studies

1. 1. Seminal plasma acid phosphatase from the mini Rock cocks was purified on a Sepharose 6B column and was not homogenous on polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Its stability in time was also determined. 2. 2. In the same time, the enzyme was crystallized in both ethanol and ammonium sulfate and this is also an evidence that this acid phosphatase was obtained in an advanced grade of purification but is present in a complex with some quantities of other proteins. 3. 3. It was inactivated by iodoacetate to a degree consistent with the modification of an active site residue. DTNB and thiosulfate also inhibited this enzyme. 4. 4. The enzyme was sensitive to 6 M guanidinum hydrochloride which in the presence and absence of 0.25 M mercaptoethanol produces a deep loss of activity. After dialysis the activity was increased during the first 10 days up to 66.2% of the initial one. 5. 5. In the presence of molar concentration of mercaptoethanol, the enzyme activity is deeply decreased, but it is partially restored after 120hr when 1 μM CuCl 2 is added.

Purification and characterization of acute phase rat plasma thiostatin.

Thiostatin was purified from acute phase plasma of turpentine-treated rats by a novel, single-step carboxymethyl-papain Sepharose 4B column chromatographic procedure. Purified thiostatin appeared as a single band in SDS-PAGE with an estimated molecular weight of 68,000. Western blot with polyclonal rabbit anti-thiostatin IgG confirmed a homogeneous immuno-reactive 68 kDa species. Specific activity, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.972 mg kininogen equivalent per mg protein. The yield of thiostatin exceeded 60% and the protein was purified 10.7-fold.

Fluorescent Activity Assay of the Enzymes Hydrolyzing Acidic Polysaccharides

Based on the amide linkage formation catalyzed by water-soluble carbodiimide, EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), various acidic polysaccharides could be labeled by the fluorescent probe of EDAN (N-1-ethylenediaminonaphthalene). The pectinase reaction was evaluated by the EDC-OPA (o-phthalaldehyde) method [Anal. Biochem., 189, 122–125 (1990)]. Moreover, paper chromatographic purification of the glucuronic acid-EDAN adduct allowed a 57-fold increase in the sensitivity of detection limit, i.e., about 0.19 nmol of glucuronate could be measured. Pectin–EDAN adduct worked as the substrate for pectinase and changes in the molecular weight distribution during the course of pectinase digestion were monitored by gel filtration with a Sepharose 6B column. In the case of hyaluronic acid–EDAN adduct, the fluorescent substrate was unfavorable to the hyaluronidase action and 2 to 5-fold higher Kms were obtained with the hyaluronic acids of lower and higher contents of EDAN, respectively.

Two lectins from the marine sponge Halichondria okadai. An N-acetyl-sugar-specific lectin (HOL-I) and an N-acetyllactosamine-specific lectin (HOL-II).

Two lectins (HOL-I and HOL-II) were isolated from the marine sponge Halichondria okadai by affinity chromatography on a bovine submaxillary mucin (BSM)-Toyopearl and an acid-treated Sepharose 4B columns, respectively. In hemagglutination inhibition assays, GlcNAc, GalNAc, and their methyl glycosides were the most potent inhibitors among the monosaccharides tested against the HOL-I-mediated hemagglutination, suggesting that HOL-I can especially recognize the N-acetyl groups of the sugars. This N-acetyl specificity was supported by 1H NMR analyses; the highest field-shifts of the signal of the N-acetyl group among all the signals in Me beta GlcNAc were observed in 1H NMR spectra of mixtures of HOL-I and the sugar. Among the oligosaccharides tested, GlcNAc beta 1-->4(GlcNAc beta 1-->2)Man alpha 1-O(CH2)2 CH3 was the most potent inhibitor, and the inhibitory potency of the oligosaccharide was 2(4) times greater than those of GlcNAc and GalNAc. On the other hand, N-acetyllactosamine (Gal beta 1-->4GlcNAc) and its analogs were the strongest inhibitors toward HOL-II-induced hemagglutination. The agglutination was completely inert to Gal beta 1-->3GlcNAc, Gal beta 1-->6GlcNAc, Gal beta 1-->3GalNAc, Gal beta 1-->4GalNAc, and Gal beta 1-->6GalNAc. Furthermore, HOL-II exhibited no binding ability to BSM, asialo-BSM, fetuin, asialofetuin, alpha 1-acid glycoprotein, and human transferrin. These results indicate that HOL-II strictly recognizes simple Gal beta 1-->4GlcNAc unit.

Purification and properties of a membrane-bound lytic transglycosylase from Escherichia coli

A membrane-bound lytic transglycosylase (Mlt) has been solubilized in the presence of 2% Triton X-100 containing 0.5 M NaCl from membranes of an Escherichia coli mutant that carries a deletion in the slt gene coding for a 70-kDa soluble lytic transglycosylase (Slt70). The enzyme was purified by a four-step procedure including anion-exchange (HiLoad SP-Sepharose and MonoS), heparin-Sepharose, and poly(U)-Sepharose 4B column chromatography. The purified protein that migrated during denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band corresponding to an apparent molecular mass of about 38 kDa is referred to as Mlt38. Optimal activity was found in buffers with a pH between 4.0 and 4.5. The enzyme is stimulated by a factor of 2.5 in the presence of Mg2+ at a concentration of 10 mM and loses its activity rapidly at temperatures above 30 degrees C. Besides insoluble murein sacculi, the enzyme was able to degrade glycan strands isolated from murein by amidase treatment. The enzymatic reaction occurred with a maximal velocity of about 2.2 mg/liter/min with murein sacculi as a substrate. The amino acid sequences of four proteolytic peptides showed no identity with known sequences in the data bank. With Mlt38, the number of proteins in E. coli showing lytic transglycosylase activity rises to three.

The state of association of Band 3 of the human erythrocyte membrane: evidence of a hexamer

Band 3 of the human erythrocyte membrane was isolated from 32P-labelled erythrocytes in nonaethyleneglycol n-dodecyl ether (C 12E 9), Triton X-100, and Brij 58 solutions, and its states of association were studied by sucrose gradient sedimentation and by Sepharose 4B column chromatography. The sedimentation and elution profiles indicated that Band 3 exists under two stable forms in each detergent solution, a slow form and a fast form. The fraction of the fast form in a Brij 58 solution was 2–3-times higher than those of the fast forms in C 12E 9 and Triton X-100 solutions. Moreover, depending of the conditions of isolation, only the slow form or the fast form was present in a Brij 58 solution. The apparent values of sedimentation coefficients, Stokes radii, and effective masses of the slow and fast forms in C 12E 9, Triton X-100, and Brij 58 solutions were determined. On the basis of these values, we have concluded that the slow and fast forms of Band 3 were dimer and tetramer in C 12E 9 and Triton X-100 solutions but were dimer and hexamer in a Brij 58 solution.

Expression of human thyroid peroxidase in insect cells using recombinant baculovirus

The baculovirus expression system was used to overexpress recombinant human thyroid peroxidase. Sf-9 cells infected with the recombinant virus AcMNPV-hTPO synthesized hTPO protein (hTPO-bac) immunogenic on Western blots when probed with either rabbit anti-TPO peptide sera or pooled human anti-TPO sera (MS12/89). hTPO-bac was a major constituent of the membrane fraction from the infected cells, constituting 14.9% and 10.1% of the 1% deoxycholate-soluble and insoluble fractions, respectively, as judged by densitometry. Recombinant hTPO-bac was extracted from cellular membranes with 1% deoxycholate and partially purified by Sepharose 6B column chromatography. Specific immunoreactivity of MS12/89 to hTPO-bac on microtiter plates was seen using ELISA. Detergent extract from wild-type virus-infected Sf-9 cells was used as background control antigen; no specific reactivity to either hTPO-bac or control antigen was seen with control sera. To determine antigenic potency, MS12/89 was incubated with increasing concentrations of various preparations of hTPO antigen and with ovalbumin as control. The capacity of the partially purified hTPO-bac to immunoneutralize human anti-hTPO standard at 50% inhibition of binding was 0.01 U/μg hTPO-bac (NIBSC Units), compared with 0.5 U/μg and 0.06 U/μg for natural hTPO and CHO-hTPO, respectively. When ELISA was performed using clinical samples of human sera to detect hTPO autoantibodies, results using hTPO-bac correlated well with those using hTPO from Graves' thyroid tissue ( r = 0.85, p = 0.02) and those using recombinant hTPO from Chinese hamster ovary cells (hTPO-CHO) ( r = 0.85, p = 0.02). These data demonstrate that recombinant hTPO-bac is reactive with human autoantibodies and can be produced in large quantities in a baculovirus expression system.

Purification and some properties of Phaseolus mungo lectin

Phaseolus mungo lectin in pure form was isolated from seeds by affinity chromatography on a galactosyl Sepharose 6B column. The lectin contained 8.3% neutral carbohydrate. It absorbed maximally near 278 nm, and the corresponding maxima in fluorescence excitation and emission spectra occurred, respectively, at 278 and 337 nm. Its elution profile from an analytical Sephadex G-150 column was consistent with a molecular weight of 137 000 and a Stokes radius of 4.3 nm; the frictional ratio, f/f0, of the lectin was calculated to be 1.26. The lectin moved as a single protein band of Mr 66 000 in SDS-PAGE under both reducing and nonreducing conditions. The results suggested that the lectin is a dimer whose overall native conformation is nearly globular. From the effect of 10 saccharides on the hemagglutinating activity of lectin against trypsinized rabbit erythrocytes, it was concluded that the lectin was galactose/N-acetylgalactosamine-specific, having significantly reduced affinity for N-acetylgalactosamine. The lectin showed preference for alpha-glycoside and probably does not contain a hydrophobic binding site.

Purification and characterization of high molecular weight (HMW) and low molecular weight (LMW) immunoglobulins from the serum of the dogfish, Scyliorhinus canicula, and the identification of antibody forming tissues

1.1. High molecular weight (HMW) and low molecular weight (LMW)-like Ig was purified and characterized from dogfish, Scyliorhinus canicula serum.2.2. HMW Ig was isolated by gel filtration on a Sepharose 6B column and by fractionation on an anion exchange Mono Q FPLC column. The LMW-Ig was separated on a rabbit anti-dogfish Ig immunoaffinity column and a Zorbax G-250 HPLC gel filtration column.3.3. Both LMW and HMW-Ig forms had identical heavy and light chains of 70 kDa and 25 kDa respectively on reducing SDS-PAGE. Intact HMW-Ig had a Mr similar to pentameric human IgM of 952 kDa, while the LMW-Ig was 160–200 kDa as estimated by non-reducing SDS-PAGE.4.4. Anti-keyhole limpet haemocyanin serum was raised in dogfish and fractionated on a gel filtration Sepharose 6B column. The immunoreactivity fractions corresponded to the HMW-Ig peak with a Mr of ca 950 kDa.5.5. Specific antiserum was raised against dogfish HMW-Ig in rabbits and employed to identify antibody secreting cells in the spleen, Leydig and epigonal organs of the adult dogfish.

Removal of beta III isotype enhances taxol induced microtubule assembly.

The interaction of beta III-depleted tubulin with taxol was investigated. A monoclonal antibody against the beta III tubulin isotype was immobilized on a sepharose 4B column and used to remove the beta III tubulin isotype from unfractionated tubulin. The assembly of beta III-depleted tubulin in the presence of taxol was enhanced compared to unfractionated tubulin. The critical concentration of unfractionated tubulin in the presence of 10 microM taxol is 0.4 mg/ml, while the critical concentration of beta III-depleted tubulin is 0.16 mg/ml. At different concentration of taxol, the assembly of beta III-depleted tubulin is increased relative to that of unfractionated tubulin and reaches the maximum at about a 1:1 ratio of tubulin and taxol. The assembly of unfractionated tubulin and beta III-depleted tubulin has also been studied by electron microscopy. After 2 minutes at 37 degrees C, unfractionated tubulin assembly in the presence of 10 microM taxol results only in ribbon-like and ring structures; there are no visible microtubules. By 5 minutes, microtubules appear and increase in length. The assembly of beta III-depleted tubulin in the presence of 10 microM taxol occurs more quickly. In contrast to the case with unfractionated tubulin, beta III-depleted tubulin assembles within 2 minutes into microtubules which increase in length with time. At 30 minutes, microtubules assembled from beta III-depleted tubulin are shorter than the microtubules assembled from unfractionated tubulin. There is no visible difference between the microtubules assembled from unfractionated tubulin and beta III-depleted tubulin. Taxol-induced beta III-depleted tubulin assembly is more resistant to the inhibiting effect of podophyllotoxin and colchicine. It is also less sensitive to the inhibiting effect of cold temperature.

Purification of human neutrophil collagenase, establishment of its monoclonal antibodies and application to gingival crevicular neutrophils

In order to investigate the role of neutrophil collagenase in the periodontal disease, human neutrophil collagenase was purified and two monoclonal antibodies against this enzyme were obtained. This enzyme was purified by four step-affinity chromatography: heparin-aminocellurofine, gelatin Sepharose 4B, collagen-Sepharose and collagenase inhibitor column chromatographies. To produce the monoclonal antibody against the enzyme, the Balb/c mouse was immunized and its spleen cells were fused with the mouse myeloma cells. Two monoclonal antibodies to the enzyme, 2F3 (IgG1) and 3F12 (IgG1), which recognized a conformational structure of the enzyme apart from its catalytic site were obtained. Both antibodies were monospecific to leukocyte collagenase and did not cross-react with the other metalloproteinases such as leukocyte gelatinase, skin fibroblast collagenase, gelatinase and stromelysin. Using these monoclonal antibodies, collagenase was stained granularly in gingival crevicular neutrophils.

Purification and Characterization of a Novel Esterase That Hydrolyzes Cannabinoid-11-hydroxyesters from Mouse Hepatic Microsomes

A novel membrane-bound esterase was purified from hepatic microsomes of male ddN mice using ω-aminooctyl Sepharose-4B, DEAE-5PW, CM-Sephadex C-50 and/or hydroxylapatite column chromatographies. The purified enzyme (ES46.5K) showed a single band on a sodium dodecylsulfate-polyacrylamide gel with a mimimum molecular weight of 46.5 Kd. The N-terminal amino acid sequence of the enzyme had no homology to those of known carboxylesterases. ES46.5K exhibited esterase activities toward 11-acetoxy derivatives of tetrahydrocannabinol (THC), whereas the enzyme did not hydrolyze phenolic acetates of cannabinoids (Δ8-THC, Δ9-THC and cannabidiol) except for 1-acetyl-cannabinol. ES 46.5K also possessed esterase activity toward p-nitrophenylacetate, 1-naphtylacetate and 2-acetylaminofluorene. Emulgen 911 and deoxycholate affected differently on the esterase activity of ES46.5K and carboxylesterase purified from porcine liver. Immuno-reactive protein to antibody against ES46.5K was present in hepatic microsomes from mice and rabbits, but not those from rats, guinea pigs and monkeys. The present study demonstrates that mouse hepatic microsomes contain a novel type of esterase having molecular weight of 46.5 Kd and high catalytic activity toward 11-hydroxy-esters of cannabinoids.

Conformation of group “a” epitope in hepatitis B surface antigen

To elucidate structure of group “a” epitope, mouse antibodies that express idiotype monoclonal antibody and anti-idiotype monoclonal antibody against the group-specific “a” determinant were purified by hydroxyapatite column. To obtain hepatitis B surface antigens (HBsAg), HBsAg positive blood was sequencially purified by ammonium sulfate precipitation, hydroxyapatite, sepharose 4B column chromatography and ultracentrifugation. The major protein (p25) and glycoprotein (gp30) of HBsAg were, isolated by concanavalin-A sepharose 4B. The ability of p25-gp30 among the HBsAg to inhibit the idiotype-anti-idiotype reaction was dependent on conformation, since reduced and alkylated p25-gp30 virtually lost their inhibitory capacity when compared to native HBsAg. The data suggest that hepatitis B antigen is a conformational antigen critically dependent upon the disulfide bonds of p25-pg30.

Putative apolipoprotein B-100 in the freshwater turtle Chrysemys picta: effects of estrogen and progesterone

1. 1. The isolation and purification of a putative apolipoprotein B-100 in the plasma of the freshwater turtle Chrysemys picta is described. 2. 2. The protein was purified through differential ultracentrifugation and subsequent Sepharose 6B column chromatography. 3. 3. The molecular weight of the protein determined by electrophoresis was approximately 350 kDa. 4. 4. An antibody to chicken apolipoprotein B-100 specifically recognizes this 350 kDa protein in Western blots, suggesting its identity with apolipoprotein B-100. 5. 5. An antibody to the putative Chrysemys apolipoprotein B-100-like protein was developed and used in an ELISA to quantitate protein levels in plasma. 6. 6. Acute estrogen treatment increased levels of apolipoprotein B-100 ( 7.64± 0.79 mg/ml plasma ) over that of control animals ( 5.07 ± 1.74 mg/ml plasma ). 7. 7. In contrast, chronic estrogen treatment reduced apolipoprotein B-100 significantly to 2.94 ± 0.53 mg/ml plasma ( P < 0.05).

Determination of the Antigenic Protein Size Associated with Faba Bean Phyllody MLO by Using (SDS-PAGE) Electrophoresis and Immunotransfer

The antigenic proteins of MLO associated with faba bean phyllody (FBP) occurring in the Sudan were studied by using SDS-PAGE electrophoresis followed either by silver nitrate staining or by transferring to nitrocellulose membrane probed with specific polyclonal antiserum. No remarkable differences between healthy and FBP infected plants were observed when the gel was colored with silver nitrate. In contrast, after probing the transferred membrane with the specific polyclonal FBP antisera, band formation was only detected with FBP infected plants. These results were treated through an image analyser using a BIOLAB logicial. The analysed proteins measure approximately 18,000 and 36,000 daltons with regard to the protein molecular weight niarkers used (Bio-Rad). Possibility of the existence of a dimer is discussed. The localization of the bands is the same whatever the origins of FBP: Vicia faba or Crotalaria saltiana. However, the partial purification of the MLO including differential centrifugations followed by precipitation with polyethylene glycol (PEG) and passage through a column of Sepharose 4B were found to be essential for having clear electrophoretic profiles.

Purification and properties of aminopeptidase H from chicken skeletal muscle.

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.