Showing papers on "Affinity chromatography published in 1979"

Identification of Ia glycoproteins in rat thymus and purification from rat spleen.

Abstract: A fraction of la-like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody-secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la-like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I-A subregion of the H-2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti-la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la-positive B lymphocyte, and 45 000 molecules per la-positive thymocyte. From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.

Purification of fibronectin from human plasma by affinity chromatography under non-denaturing conditions.

Abstract: Fibronectin was purified from human plasma by affinity chromatography under nondenaturing conditions. The method was based on the previously known binding of fibronectin to gelatin. The novel features of our method are the use of arginine in the elution of fibronectin from immobilized gelatin [Vuento & Vaheri (1978) Biochem. J. 175, 333-336] and the use of arginine-agarose as second affinity step. The purified protein was homogeneous as judged by polyacrylamide-gel electrophoresis, analytical ultracentrifugation and two-dimensional immunoelectrophoresis. The yield was 60%. We propose that the method would be useful in preparation of fibronectin for studies on its biological activities, where it is important that the protein is obtained in a native state.

Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein.

Abstract: A glycoprotein with affinity for the Fc region of immunoglobulin was isolated from extracts of cultured cells infected with herpes simplex virus type 1, and experiments were done to characterize its properties and to investigate whether it could account for the Fc-binding activity previously demonstrated on the surfaces of intact herpes simplex virus-infected cells. The technique of affinity chromatography was used to identify and isolate the Fc-binding glycoprotein and to demonstrate the specificity of its interaction with immunoglobulin G-Fc. Although three electrophoretically distinguishable Fc-binding polypeptides were identified by affinity chromatography, these three species appear to be different forms of the same translation product, based on comparisons of proteolytic digestion products and on the kinetics of appearance of each form after a brief pulse with radioactive amino acids. The results suggest that one polypeptide, designated pE, is processed to yield gE1, which is in turn processed to yield gE2. Both gE1 and gE2 are glycosylated membrane proteins and both can be labeled by the lactoperoxidase-catalyzed radioiodination of intact infected cells, indicating the presence of these proteins in surface membranes of the cells. Increases in the amounts of gE1 and gE2 at the cell surface were found to parallel the increase in Fc-binding activity of intact infected cells.

Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes

Abstract: A leukocyte-common (L-C) antigen which can be dominant as an immunogen in rabbit anti-rat thoracic duct lymphocyte serum has been purified from rat thymocytes. Initially, an antigenic fragment of 100,000 apparent mol. wt. was prepared at 400 to 900-fold purification by lentil lectin affinity chromatography and gel filtration in deoxycholate. Mice were then immunized with this fraction, and a hybrid myeloma cell line secreting antibody to the L-C antigen was prepared by cell fusion. This antibody was used for affinity chromatography and gave pure L-C antigen at 1400-fold purification compared with thymocytes. The L-C antigen is a major membrane glyco-protein of rat thymocytes and has an apparent mol. wt. of 150,000 as determined by electrophoresis on polyacrylamide gels in sodium dodecyl sulfate. The antigen constitutes one of the three thymocyte glycoproteins which stain intensely for carbohydrate with periodic acid Schiff stain. It is present on greater than 95% of thymocytes, bone marrow cells and thoracic duct lymphocytes.

Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP

Abstract: A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.

Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by 3-methylcholanthrene or phenobarbital.

Abstract: Glucuronidation reactions catalysed by rat liver microsomal UDP-glucuronyltransferase are differentially inducible by 3-methylcholanthrene and phenobarbital. To elucidate the molecular basis of this functional heterogeneity the enzyme was purified from livers of rats pretreated with the inducing agents. Using cholate solubilization, chromatography on Bio-Gel A-1.5m and on DEAE-cellulose in the presence of the nonionic detergent Brij 58, two enzyme forms could be separated. Both forms were subsequently purified to apparent homogeneity by affinity chromatography on UDP-hexanolamine Sepharose 4B. 3-Methylcholanthrene-inducible enzyme activity towards 1-naphthol, 4-nitrophenol, 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine copurified with one enzyme form (enzyme 1). In contrast phenobarbital-inducible enzyme activity towards morphine, chloramphenicol and 4-hydroxybiphenyl was associated with the other enzyme fraction (enzyme 2). Sodium dodecylsulfate/polyacrylamide gels showed similar molecular weights of 54000 for enzyme 1 and 56000 for enzyme 2. The results suggest the presence of at least two forms of UDP-glucuronyltransferase in rat liver. Factors affecting enzyme activity in purified and membrane-bound states are discussed.

A structural basis for four distinct elution profiles on concanavalin A – Sepharose affinity chromatography of glycopeptides

Abstract: Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanavalin A (Con A) – Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains:Such glycopeptides have only a single mannose residue capable of interacting with Con A – Sepharose; an interacting mannose residue is either an α-linked nonreducing terminal residue or an α-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl α-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures:where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A – Sepharose and elution as a sharp peak with 0.1 M methyl α-D-glucopyranoside; glycopeptides giving this pattern h...

Enzymatic activities associated with a purified simian virus 40 T antigen-related protein.

Abstract: A protein antigenically related to the simian virus (SV 40) A gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2+D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + Pi at a rate of 3 μmol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.

Resolution of adenylate cyclase sensitive and insensitive to Ca2+ and calcium-dependent regulatory protein (CDR) by CDR-Sepharose affinity chromatography

Abstract: Partially purified adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from bovine brain cortex was fractionated into two separate forms by calcium-dependent regulatory protein (CDR)-Sepharose affinity chromatography. The major form of the enzyme, comprising approximately 80% of the applied activity, did not bind to the affinity column in the presence of Ca2+ and was insensitive to the CDR. Approximately 20% of adenylate cyclase activity was absorbed to CDR-Sepharose in the presence of Ca2+. This activity was stimulated by Ca2+ and CDR. This study directly demonstrates that brain cortex contains Ca2+-CDR-sensitive and -insensitive forms of adenylate cyclase and indicates that CDR-Sepharose may be a useful tool for purification of adenylate cyclase. The Ca2+ -stimulated adenylate cyclase was purified at least 55-fold with a 13% yield.

Characterization of a histone-like protein extracted from yeast mitochondria

Abstract: Analysis of proteins isolated by affinity chromatography on DNA-cellulose from highly purified yeast mitochondria shows that these organelles do not contain histones but have in abundance a DNA-binding protein of 20,000 daltons. The purification yield of this protein, called HM, indicates that mitochondria have at least an equal mass of HM relative to DNA. The amino acid composition and its electrophoretic characterization reveal that HM, rich in lysine, is slightly basic and heat stable. HM appears to be coded by the yeast nucleus, as shown by its presence in several "petite" mutants. We have shown that HM, like histones or histone-like proteins, is able to introduce superhelical turns into circular relaxed DNA in the presence of a nicking-closing activity.

Purification of Streptomyces chromofuscus Phospholipase D by Hydrophobic Affinity Chromatography on Palmitoyl Cellulose

Abstract: Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3.1.4.4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5.1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1.43 mM for lecithin, 100% and 1.67 mM for lysolecithin, and 22% and 0.56 mM for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+. Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity. Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability: albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; all the lipids lowered the heat-stability. The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation. Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability.

C1q: Isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay

Abstract: A C1q-depleted reagent (C1qD) was obtained from the nonadsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 × 1013 effective molecules/mg and 0.5 to 1 × 1012 effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.

A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum.

Abstract: A procedure is described which permits the rapid isolation of large amounts of elastase and cathepsin G from purulent sputum. This procedure involves: (1) digestion of sputum with DNase, (2) extraction of the insoluble residue that remains with 1 M NaCl, pH 8, (3) affinity chromatography on Sepharose-bound Trasylol, and (4) separation of the two enzymes by chromatography on CM-Sephadex. Starting with 500 g of sputum it was possible to isolate 175 mg of each of these two enzymes within 7 to 10 days. Active site titration indicated both enzymes to be at least 97% pure. Disc gel electrophoresis in the presence and absence of SDS and amino acid sequence of the N-terminal region support the conclusion that the elastase and cathepsin G isolated from sputum a re identical to the same enzymes isolated directly from the leukocytes of human blood.

Vertebrate lectins, Comparison of properties of beta-galactoside-binding lectins from tissues of calf and chicken.

Abstract: Beta-galactoside-binding lectins were isolated from various calf tissues and from chicken hearts by affinity chromatography on asialofetuin-Sepharose, and were compared with respect to biochemical characteristics, binding properties, antigenic cross-reactivity, and cellular localization. The lectins are all thiol group-requiring, divalent cation-independent dimers, of apparent monomer mol wt 12,000 (calf lectins) or 13,000 (chicken lectin), and acidic pI. The calf lectins appear essentially identical by dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid composition, and radioimmunoassay, while the chicken lectin is distinctly different by these criteria. However, all of the lectins competed for the same binding sites on rabbit erythrocytes, and could be inhibited by the same saccharide haptens (notably lactose and thiodigalactoside). Immuno-fluorescence studies on several cultured cell lines revealed that the bovine and chicken lectins had primarily an intracellular cytoplasmic localization. The beta-galactoside-binding lectins of vertebrates appear to be species-specific rather than tissue-specific.

Isolation of an aminoglycoside receptor from guinea pig inner ear tissues and kidney.

Abstract: Affinity chromatography was used to isolate aminoglycoside receptors from inner ear tissues and kidney. Neomycin was immobilized on glass beads and served as the stationary phase in column chromatography. Fractionation of an organic tissue extract on this matrix demonstrated two components with high affinity for neomycin: phosphatidyl inositol phosphate and phosphatidyl inositol diphosphate. The toxicity of aminoglycosides is explained on the basis of a drug-interaction with these lipids.

Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart.

Abstract: The calcium-dependent regulatory protein (CDR).Ca2+ sensitive cyclic nucleotide phosphodiesterase was purified to apparent homogeneity from bovine heart by using ammonium sulfate fractionation, DEAE-ceelulose chromatography, and CDR-Sepharose affinity chromatography. The enzyme was purifed 13 750-fold with a 10% yield and a specific activity of 275 mumol of cAMP min-1 mg-1. The purified enzyme ran as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 57 000. Phosphodiesterase activity was stimulated 10-fold by Ca2+ and CDR with half-maximal activation occurring at 9 ng/assay. [125I]CDR was cross-linked to the purified phosphodiesterase by using dimethyl suberimidate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked products revealed a number of discrete 125I-labeled bands. The molecular weights of the cross-linked products indicate that the stoichiometry of the phosphodiesterase complex is A2C2, where A is the phosphodiesterase catalytic subunit and C is the calcium-dependent regulatory protein.