Showing papers on "Affinity chromatography published in 1983"

Monoclonal antibodies: Principles and practice : production and application of monoclonal antibodies in cell biology, biochemistry, and immunology

Abstract: Introduction. The Antibody Response. Cellular Basis of the Immune System. Nature of Antigens. Antibody Structure and Function. Genetics of Antibodies. Introduction to Monoclonal Antibodies. Production of Monoclonal Antibodies. Purification. Fragmentation and Isotopic Labeling of Monoclonal Antibodies. Analysis of Antigens Recognized By Monoclonal Antibodies. Affinity Chromatography Immunofluorescence. Immunohistology. Construction, Screening and Expression of Recombinant Antibodies. Generation of Conventional Antibodies.

Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions.

Abstract: Immobilized metal ion affinity adsorption (IMA adsorption) is a collective term that is proposed to include all kinds of adsorptions whereby metal atoms or ions immobilized on a polymer cause or dominate the interaction at the sorption sites. IMA chromatography is one of the most powerful methods available to date for protein fractionation although this is not as yet widely recognized. This study deals with the theoretical aspects of IMA adsorption and its practical applications as exemplified by the various results reported here. The synthesis of iminodiacetate-substituted agarose (IDA-agarose) and tris(carboxymethyl)ethylenediamine-agarose (TED-agarose) is described. Many types of metal ions can easily be immobilized on these gel derivatives to form IMA adsorbents. We have not observed any damage to the proteins during the adsorption-desorption process. After performance of an experiment, the gels can easily be regenerated and can be loaded with the same or a different metal ion for an ensuing experiment. Specific adsorption is demonstrated for serum proteins on immobilized Ni(II) and Fe(III). Ligand-specific desorption (affinity elution) is also demonstrated by including in the buffer system certain solutes which are similar to or identical with some particular amino acids found in proteins. High concentrations of certain salts that affect the structure of water, such as Na2SO4, promote coordinate covalent bonding of proteins by a mechanism that is apparently similar to that found in hydrophobic interactions. Neutral detergents and aquoorganic solvents may be used. This opens up the possibility for the fractionation of membrane components. The IMA-adsorption method could also be expanded to other areas besides protein fractionation.

Serum spreading factor (vitronectin) is present at the cell surface and in tissues

Abstract: Monoclonal antibodies were prepared against a cell attachment-promoting protein, serum spreading factor, which had been partially purified from human serum by chromatography on glass bead columns. The antibodies selected were those that reacted with polypeptides that had cell attachment-promoting activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunochromatography of human plasma on columns containing the monoclonal antibodies followed by affinity chromatography on heparin-Sepharose yielded material that in sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis gave polypeptides of molecular mass 65 and 75 kilodaltons. Both polypeptides bound each of three monoclonal antibodies and had cell attachment-promoting activity after transfer to nitrocellulose filters. Immunofluorescent staining of tissues with the monoclonal antibodies revealed a fibrillar pattern that was mostly associated with loose connective tissue and overlapped with fibronectin fibrils. Fetal membrane tissue, which showed strong staining with the antibodies in immunofluorescence, also gave 65- and 75-kilodalton polypeptides with cell attachment-promoting activity after chromatography on columns containing the monoclonal antibodies. One source of the tissue protein may be fibroblastic cells, because cultured human fibroblasts also stained with the monoclonal antibodies. The staining was fibrillar and appeared to be associated with the cell surface extracellular matrix. We propose the name "vitronectin" for the various forms of this protein, on the basis of its binding to glass and its adhesive properties.

Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells.

Abstract: We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix.

Tyrosine-specific protein kinase activity is associated with the purified insulin receptor

Abstract: Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr 135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies. When this preparation was incubated with [gamma-32P]ATP in the presence of MnCl2 (2 mM) and analyzed in NaDodSO4/acrylamide gel electrophoresis, only the Mr 95,000 band was labeled. Preincubation with several concentrations of insulin increased the 32P incorporation into this peptide in dose-dependent fashion, whereas insulin-like growth factors were approximately equal to 2% as potent and epidermal growth factor had little or no effect, consistent with their known affinities for the insulin receptor. Insulin stimulation of phosphorylation of the Mr 95,000 subunit of the receptor was observed also in immunoprecipitates of this highly purified insulin receptor by anti-insulin-receptor antibodies. Phosphoamino acid determination revealed only phosphotyrosine in both the basal and insulin-stimulated states. These data suggest that a tyrosine-specific protein kinase activity is closely associated with insulin receptor, and this may be important in the signal transmission required for insulin action.

Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum.

Abstract: The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.

Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells

Abstract: We have developed a method for purifying L-CAM, the cell adhesion molecule from embryonic chicken liver cells, and have compared its properties with those of N-CAM, the neural cell adhesion molecule. L-CAM was released from membranes with trypsin, purified by a series of chemical techniques, and used to generate monoclonal antibodies which allowed the identification of the intact L-CAM molecule from membranes. The monoclonal antibodies were used to isolate trypsin-released L-CAM in a single step by affinity chromatography. Material purified by either technique was predominantly a component of Mr 81,000 on NaDodSO4/polyacrylamide gel electrophoresis with a pI of 4.0-4.5. Rabbit antibodies to this component and to the Mr 81,000 species that had been further purified on NaDodSO4/polyacrylamide gel electrophoresis displayed all of the activities of anti-L-CAM. Some of the trypsin-released L-CAM bound specifically to lentil lectin, suggesting that L-CAM is a glycoprotein. The apparent molecular weight of material having L-CAM antigenic determinants depended upon the procedures used to extract membranes; this appears to account for the various values reported previously in the literature. Both the rabbit serum antibodies and the monoclonal antibodies detected the Mr 81,000 species on immunoblots of unfractionated trypsin-released material. Immunoblots of whole liver cell membranes with the same antibodies revealed a major Mr 124,000 component, with minor components of Mr 94,000 and 81,000. Active L-CAM derivatives released by trypsin in the presence of EGTA were detected as a species of Mr 40,000. L-CAM derivatives obtained by extraction of membranes with EDTA alone appeared as species of Mr 53,000, 62,000, and 81,000. The combined results suggest that L-CAM on the cell surface is an acidic glycoprotein of Mr 124,000. In the presence of calcium, the molecule can be released from membranes by trypsin as a soluble Mr 81,000 fragment; in the absence of calcium, it is released by either endogenous proteases or by trypsin as a variety of smaller fragments.

A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates Synapsin I: partial purification and characterization

Abstract: A calcium/calmodulin-dependent protein kinase, which phosphorylates a synaptic vesicle-associated protein designated Synapsin I, has been shown to be present in both soluble and particulate fractions of rat brain homogenates. In the present study, the particulate activity was solubilized by washing with a low ionic strength solution, and the enzymes from the two fractions were partially purified by ion exchange chromatography and calmodulin-Sepharose affinity chromatography. By each of several criteria, the partially purified enzymes from the two sources were indistinguishable. These criteria included specificity for various substrate proteins, concentration dependence of activation by calcium and calmodulin, pH dependence, and apparent affinities for the substrates Synapsin I and ATP. The mild conditions that released the particulate enzyme indicated that it was not tightly bound to the membrane and suggested that it may exist in a dynamic equilibrium between soluble and particulate-bound states. The partially purified enzyme preparations from both the soluble and particulate fractions contained three proteins that were phosphorylated in the presence of calcium and calmodulin, a 50-kilodalton (Kd) protein and two proteins in the 60-Kd region. When compared by phosphopeptide mapping and two-dimensional gel electrophoresis, the proteins were indistinguishable from three proteins of corresponding molecular weights that were shown by Schulman and Greengard (Schulman, H., and P. Greengard (1978) Nature 271: 478-479) to be prominent substrates for calcium/calmodulin-dependent protein kinase in a crude particulate preparation from rat brain. The 50-Kd substrate was the major Coomassie blue staining protein in both partially purified enzyme preparations. The peak of this protein coincided with that of enzyme activity during DEAE-cellulose and calmodulin-Sepharose chromatography. These results suggest that the 50-Kd phosphoprotein may be an autophosphorylatable subunit of the Synapsin I Kinase or may exist in a complex with it.

Isolation and Characterization of a Mannan-Binding Protein from Human Serum

Abstract: A serum lectin specific for mannose and N-acetylglucosamine residues was isolated from human serum to near homogeneity mainly by affinity chromatography on a column of Sepharose 4B-mannan. The lectin, called mannan-binding protein, was a glycine-rich protein with an apparent molecular size of approximately 600,000 daltons, and had a subunit structure consisting of a single component with an apparent molecular weight of 31,000. Binding of the isolated lectin to 125I-labeled mannan was dependent upon the presence of Ca2+, proportional to the protein added, and a reversible and saturable process. Scatchard plot analysis of binding data indicated the presence of a binding site with a dissociation constant of 2.3 X 10(-9) M and a maximum capacity of 4.3 pmol of 125I-labeled mannan per microgram of protein (2.6 mol of mannan per mol of the protein). The mannan-binding protein, is different from C-reactive protein (CRP) and amyloid P-component (SAP), both of which are serum components known to bind polysaccharides in the presence of Ca2+. A distinct binding activity toward mannan which did not require Ca2+ was attributed to immunoglobulins (IgG).

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes.

Abstract: A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.

Opsonizing properties of an isolated hemolymph agglutinin and demonstration of lectin-like recognition molecules at the surface of hemocytes fromMytilus edulis

Abstract: Mytilus hemolymph was found to contain an agglutinin which could be inhibited by mucin. The agglutinin was isolated by affinity chromatography using neuraminidase-treated mucin/Sepharose.

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture.

Abstract: A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.

The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle.

Abstract: Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex G-200 (Mr = 98000 +/- 4000), chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The alpha-subunit of phosphorylase kinase, protein phosphatase inhibitor-1 and the myosin P-light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the beta-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B, ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor eIF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-1. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 microM Ca2+ in the absence, and at 0.5 microM Ca2+ in the presence, of 0.03 microM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half-maximal inhibition occurred at 45 microM in the absence and 35 microM in the presence of 0.03 microM calmodulin. The metabolic role of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulin-binding protein of neural tissue termed calcineurin or CaM-BP80 [Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Lett. 137, 80-84].

Affinity-purified anti-B-50 protein antibody: interference with the function of the phosphoprotein B-50 in synaptic plasma membranes.

Abstract: Affinity-purified anti-B-50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B-50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B-50 protein and its adrenocorticotropin-sensitive protein kinase, obtained from rat brain. Anti-B-50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B-50 protein isolated by an improved procedure. The purified antibodies reacted only with the B-50 and B-60 protein, a proteolysis derivative (of B-50), as assessed by the sodium dodecyl sulfate-gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B-50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5-diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4-phosphate was not altered. Inhibition by ACTH 1–24 of the endogenous phosphorylation of B-50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-diphosphate. These data support our hypothesis on the functional interaction of B-50 protein and phosphatidylinositol 4-phosphate kinase in rat brain membranes. The evidence shows that purified anti-B-50 protein antibodies can be used to probe specifically the function of B-50 protein in membranes.

Fibronectin. A new therapy for corneal trophic ulcer

Abstract: • Fibronectin, a glycoprotein, is present in plasma and extracellular matrix and is responsible for cellular adhesion. With the use of affinity chromatography, we purified plasma fibronectin from two patients with trophic corneal ulcer and persistent epithelial defect after conventional therapy was ineffective. Reepithelialization began three days after initiation of treatment with autologous purified fibronectin eyedrops, and the epithelial defects disappeared completely within three weeks. No recurrence was observed after treatment ended. By modifying the original chromatographic procedure, we were able to obtain purified fibronectin in two hours, which allowed us to treat outpatients as well as inpatients.

Isolation and characterization of a collagen-binding glycoprotein from chondrocyte membranes.

Abstract: A collagen-binding glycoprotein was isolated from purified chick chondrocyte surface membranes by affinity chromatography on type II collagen-Sepharose. The purified glycoprotein has an apparent mol. wt. of 31,000 and binds to native chick collagen types I, II, III, V and M. Although it contains 30% carbohydrates, the majority of which is fucose, it is hydrophobic and soluble only in detergents. The integral membrane protein character of the 31-K protein became apparent from its ability to insert into lecithin vesicles. Liposome-inserted 31-K protein binds 125I-labelled type II collagen in the presence of 0.5 M NaCl, while detergent-solubilized 31-K protein is dissociated from type II collagen by 0.05-0.1 M NaCl. Electron microscopic studies employing the rotary shadowing technique indicate that 31-K protein particles bind to the ends of collagen molecules. We propose that this glycoprotein serves as anchorage site for extracellular collagen to the chondrocyte membrane and thus may be involved in cell-matrix interactions in cartilage.