Showing papers on "Affinity chromatography published in 1977"

Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution.

Abstract: The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38,000 daltons. Human CobINA was found to be a glycoprotein containing at least 10.7% carbohydrate and to have a normal serum concentration of 34 +/- 7 mug/ml (mean +/- 1 SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating beta1H were removed from these proteins with anti-beta1H Sepharose. However, in the presence of highly purified beta1H and C3bINA, both C3bIna, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their structure. The action of C3bINA and beta1H on C3b produced two fragments of the alpha1-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40,000 daltons. The action of C3bINA and beta1H on C4b resulted in cleavage of the alpha'-chain giving rise to the 150,000-dalton C4c and the 49,000-dalton C4d fragments which dissociated without reduction. To produce from C3b the immunochemically defined C3c and C3d, fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.

Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ.

Abstract: A method is described for the large-scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol α-toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor-rich membrane fragments is composed of only one class of membrane fragments covered with 8-nm rosettes identified as acetylcholine receptor molecules. This preparation is used as a starting material for the detergent solubilization and the large-scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X-100 extract of receptor-rich membranes done in the presence of 2-mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy. Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecylsulfate reveals three bands of apparent molecular weights 40000 ± 1000, 50000 ± 2000 and 66000 ± 2000, the two heaviest ones being present in significantly lower amounts than the 40000-Mr one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the 40000-Mr band to the 50000 and 66000-Mr ones.

Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3',5'-monophosphate dependent protein kinases.

Abstract: The heat-stable protein inhibitor (Walsh, D. A., et al. (1971), J. Biol. Chem. 246, 1977--1985) of the cyclic adenosine 3',5'-monophosphate dependent protein kinase has been isolated in pure form from rabbit skeletal muscle after a 430 000-fold purification with a 47% yield. The four-step procedure involves sequentially a heat treatment, batchwise anion and cation exchange, and affinity chromatography on protein kinase catalytic subunit covalently coupled to Sepharose 4B. The inhibitor is an acidic protein (pI = 4.24) of molecular weight 11 300. It contains 98 amino acid residues none of which contains sulfur and only 2 (phenylalanine and tyrosine) are aromatic. The NH2-terminus is blocked. The muscle content is ca. 0.6 mg of inhibitor per L of intracellular water. The inhibitor is tightly bound to the catalytic subunit of protein kinase (Ki congruent to 2 X 10(-9) M) and acts competitively with respect to the protein substrates. Protein kinase recognizes a short stretch of the inhibitor sequence, in which arginyl side chains play a crucial role. A study of various competitive inhibitors of the kinase confirms the importance of guanidino groups and hydrophobic side chains in the specific interaction with the substrate binding site.

The Interaction of Heparin with an Apoprotein of Human Very Low Density Lipoprotein

Abstract: An arginine-rich apoprotein obtained from human triglyceride-rich lipoprotein was isolated on a heparin affinity column when either the aqueousor urea-soluble apoproteins were applied to the column. Of all the aqueous- or urea-soluble apoproteins, only this arginine-rich protein exhibited a binding affinity to heparin. This protein was eluted from the column at sodium chloride concentrations above 0.35 M in the absence of urea and between 0.17-0.2 M when isolated in urea. The protein has been characterized by amino acid analysis, immunoelectrophoresis, dodecyl sulfate polyacrylamide electrophoresis, isoelectric focusing, and NH2-terminal analysis. It has the same amino acid composition, NH2-terminal, and molecular weight as previously described for human arginine-rich apoprotein. The triglyceride-rich lipoproteins of fasting normal humans were eluted as two fractions when applied to the heparin affinity column. A small amount was eluted in the unbound fraction and this species contained virtually no arginine-rich apoprotein. The bulk of the triglyceride-rich lipoproteins eluted in the bound fraction and contained appreciable amounts of arginine-rich apoprotein. The bound lipoproteins had more cholesterol and cholesterol ester and less triglyceride than the unbound. The isolated arginine-rich apoprotein was derivatized with phenylglyoxal with a resulting alteration of 75% of the arginine residues. This modified apoprotein did not bind to the heparin affinity column. Similar treatment of the whole triglyceride-rich lipoprotein produced a lipoprotein that was totally eluted in the unbound fraction.

Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase.

Abstract: Herpes simplex virus-induced DNA polymerase purified by published methods was found to be contaminated with many others proteins, including virus structural proteins. Thus, DEAE-cellulose and phosphocellulose chromatography were used in combination with affinity chromatography to purify DNA polymerase from herpes simplex virus type 1- and type 2-infected cells. The purified enzyme retained unique features of the herpesvirus-induced DNA polymerase, including a requirement for high salt concentrations for maximal activity, a sensitivity to low phosphonoacetate concentrations, and the capacity to be neutralized by rabbit antiserum to herpesvirus-infected cells. By polyacrylamide gel electrophoresis, the purified DNA polymerase was associated with a virus-induced polypeptide of about 150,000 molecular weight.

Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins.

Abstract: The effects of several commonly used detergents on the saccharide-binding activities of lectins were investigated using lectin-mediated agglutination of formalin-fixed erythrocytes and affinity chromatography of glycoproteins on columns of lectins immobilized on polyacrylic hydrazide-Sepharose. In the hemagglutination assays, Ricinus communis I (RCA1) and II (RCAII), concanavalin A (Con A), and the agglutinins from peanut (PNA), soybean (SBA), wheat germ (WGA), and Limulus polyphemus (LPA) were tested with several concentrations of switterionic, cationic, anionic, and nonionic detergents. It was found that increasing detergent concentrations eventually affected hemagglutination titers in both test and control samples, and the highest detergent concentrations not affecting lectin hemagglutinating activities were determined. The effects of detergents on specific binding of [3H]fetuin and asialo[3H]fetuin to and elution from columns of immobilized lectins were less severe when compared with lectins in solution, suggesting that the lectins are stabilized by covalent attachment to agarose beads. Nonionic detergents did not affect the binding efficiency of the immobilized lectins tested at concentrations used for membrane solubilization while cationic and zwitterionic detergents caused significant inhibition of Con A- and SBA-Sepharose activities. In sodium deoxycholate (greater than 1%) only RCAI-Sepharose retained its activity, whereas the activities of the other lectins were reduced dramatically. Low concentrations of sodium dodecyl sulfate (0.05%) inhibited only the activity of immobilized SBA, but at higher concentration (0.1%) and prolonged periods of incubation (16 h, 23 degrees C) most of the lectins were inactivated. These data are compared with previous reports on the use of detergents in lectin affinity chromatography, and the conditions for the optimal use of detergents are detailed.

Role of lectins in plant-microorganism interactions: I. Binding of soybean lectin to rhizobia.

Abstract: Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ( 3 H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine. The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 × 10 6 . While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth. Quantitative binding studies with 3 H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 × 10 7 m −1 . Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates.

Distribution of Neuraminidase in Arthrobacter and Its Purification by Affinity Chromatography

Abstract: Neuraminidase [sialidase, EC 3.2.1.18] was found to be widely distributed in bacteria belonging to Arthrobacter. Among these bacteria, Arthrobacter ureafaciens, A. oxydans, and A. aurescens produced relatively potent neuraminidase activities. For the production of this enzyme, not only colominic acid, a homopolymer of N-acetylneuraminic acid, but also N-acetylneuraminic acid, the reaction product of this enzyme, are effective as sources of carbon. An affinity adsorbent specific for neuraminidase was prepared by cross-linking colominic acid with soluble starch by means of epichlorohydrin. Neuraminidase from A. ureafaciens could be purified on this affinity column. The purified neuraminidase was shown to be free from protease, N-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. Aminoff's assay procedure for sialic acid was modified to avoid the centrifugation step. The modified procedure gave a higher molecular extinction coefficient.

Preparative purification of the rat mast cell chymase: characterization and interaction with granule components.

Abstract: The rat mast cell granule chymotrypsinlike enzyme was purified to homogeneity from 1 M NaCl solubilized membrane and granule-rich fractions of concentrated rat peritoneal mast cells by a preparative technique utilizing chromatography on Dowex 1, filtration on Sephadex G-75, and affinity chromatography with D-tryptophan methyl ester. Acid disk gel electrophoresis of the purified chymase disclosed a single stained band with activity being eluted from a replicate sliced gel in the same region. SDS-polyacrylamide gel electrophoresis of purified protein gave a single stained band that did not change in position with reduction and alkylation. Mast cell chymase is thus a cationic protein of 25,000 mol wt composed of a single polypeptide chain. The apparent K(m) of the chymase for BTEE was 1.5 x 10(-3) M and the V(max) was 67.8 μmol/min per mg. The enzyme was inhibited by TPCK and not by TLCK. The chymase complexed with native macromolecular rat mast cell heparin in molar ratios of 12:1 and 16:1, and complete heparin uptake occurred at a 40:1 ratio of chymase to heparin. Chymase activity was partially masked by combination with heparin in the isolated granule or experimental chymase-heparin complex, and soluble purified chymase was inhibited by concentrations of 5-HT comparable to those present in mast cells. It is therefore possible that the active site of chymase in the mast cell granule is largely masked by the combined effects of macromolecular heparin and 5-HT.

Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi.

Abstract: The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a crude extract. The final step in the purification procedure is chromatography on a 5'-AMP-Sepharose 4B affinity column which results in approximately a 50-fold purification to a final specific activity of 31 mumol of NADH oxidized min-1 (mg of protein)-1 for the NADH specific FMN reductase. The NADPH specific reductase has been purified to a final specific activity of 51 mumol of NADPH oxidized min-1 (mg of protein)-1 using a NADP agarose affinity column, which results in a 70-fold purification. Molecular weights of 30 000 and 40 000 and Km's of 4.75 X 10(-5) M NADH and 4.0 X 10(-5) M NADPH have been determined for the pure NADH and NADPH specific FMN reductases, respectively. The NADPH specific FMN reductase does not utilize NADH, while the NADH specific enzyme does dehydrogenate NADPH with a maximal velocity one-tenth of that for NADH. Separate NADH and NADPH specific FMN reductases from Photobacterium fischeri could not be demonstrated.

Magnetic ferrofluids for preparation of magnetic polymers and their application in affinity chromatography

Abstract: THE use of magnetic polymers as supports for biological molecules has created much interest1–5. The inherent advantages of such preparations are, in particular, the ease of recovery of these polymers by applying a magnetic field. Thus, when used as supports for immobilised enzymes, their easy retrieval from liquors containing colloids or undissolved solids should be of great practical value1–3. Magnetic polymers have recently been tested as an alternative to conventional radioimmunoassay technique, obviating the need for vertical rotation and for the time-consuming, multiple centrifugations required with conventional solid phase procedures4–5. In the established techniques for producing magnetic polymer particles, the attachment of the biomolecules had to follow the preparation of the specific magnetic material; furthermore most of these preparations have been non-porous, exhibiting rather poor capacity. We describe here a novel and general procedure using magnetic fluids which allows ‘post-magnetisation’ of polymers already substituted with biological molecules. The properties of such preparations have been tested primarily as affinity chromatography gels. These preparations showed unaltered biospecificity when applied in general ligand-affinity chromatography studies. The simplified separation possible due to the magnetic properties of the gels eliminates the usual centrifugation and column chromatography steps. The procedure of ‘post-magnetisation’ seems to be suitable for different polymer particles, both unsubstituted gels and those carrying immobilised ligands as affinity adsorbents or enzymes.

Quantitative serological analysis of a rabbit anti-rat lymphocyte serum and preliminary biochemical characterisation of the major antigen recognised.

Abstract: Rabbit antisera to rat thoracic duct lymphocytes were examined by absorption analysis with quantitative radioimmunoassays to determine the tissue distribution of the antigens recognised and the amount of antibody against different specificities. Seven of eight sera were remarkably specific for leucocytes and one serum was analysed in detail. In this serum about 70 per cent of the antibody was leucocyte specific and most was against determinants on thymocytes, bone marrow cells, peripheral lymphocytes, and macrophages. This specificity is referred to as the leucocyte-common antigen. Smaller amounts of antibody were specific for antigens on peripheral lymphocytes but not thymocytes, and this could be subdivided into two antigens, one present on bone marrow cells and the other not. For preliminary biochemical analysis of the leucocyte-common antigen, thymocyte membrane was solubilized in deoxycholate. The homogeneity and apparent size of the antigen was determined by gel filtration and zone sedimentation on sucrose gradients. The activity behaved as a single component with the following hydrodynamic properties: sedimentation coefficient, 5.9S; partial specific volume, 0.71 ml/g; and Stokes radius, 7.5 nm. The molecular weight (including any bound deoxycholate) was calculated to be 172,000 and the frictional ratio 2.05. All activity bound to a lentil lectin affinity column and was eluted by methyl alpha-D-glucopyranoside, suggesting that the antigen is a glycoprotein.

Isolation and partial characterization of a low molecular weight acid stable protease inhibitor from human bronchial secretion.

Abstract: An acid stable protease inhibitor was isolated from human bronchial secretion. Two important stages of the purification procedure were affinity chromatography on trypsin bound to Affi-Gel 10 and ion-exchange chromatography on SP-Sephadex C-50. The isolated inhibitor appeared as a single band on analytical disc electrophoresis and eluted as a homogeneous protein peak on gel filtration on Sephadex G-75 corresponding to a molecular weight of about 10500. Amino acid analyses showed no tryptophan or histidine and as N-terminal amino acid tyrosine. No glucosamine or galactosamine was detected. The results of the analyses suggest that the purified inhibitor is identical to the low molecular weight trypsin-chymotrypsin inhibitor of human seminal plasma (HUSI-I).