Showing papers on "Affinity chromatography published in 1989"

Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton.

Abstract: We have previously reported the production of monoclonal antibodies directed against phosphotyrosine, which is the modification induced by many oncogene products and growth factor receptors. One of these antiphosphotyrosine antibodies (py20) was used in affinity chromatography to isolate phosphotyrosine (PY)-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEFs). Mice were immunized with these PY-proteins for the production of monoclonal antibodies to individual substrates. Fifteen antibodies were generated in this way to antigens with molecular masses of 215, 76, 60, and 22 kD. Antibodies to individual substrates were used to analyze the subcellular location in normal and RSV-CEFs. Antibodies to the 215- and 76-kD antigen stained focal contacts when used in immunofluorescence microscopy while anti-22-kD protein antibodies resulted in punctate staining concentrated in the margins of cells and in parallel arrays. Both distributions were altered in transformed cells. When cells were extracted with nonionic detergent under conditions that stabilize the cytoskeleton, 50% of the 76-kD protein and greater than 90% of the 22-kD protein fractionated with the cytoskeleton. This study offers a new approach to both the identification of membrane skeletal proteins in fibroblasts and changes that occur upon transformation by an activated tyrosine kinase.

Soluble cytokine receptors are present in normal human urine.

Abstract: Affinity chromatography of crude human urinary proteins on either human rIL-6, human rIFN-gamma, or anti-IFN-gamma-R mAb yielded the two respective soluble receptors in significant quantities. A single sequence of 30 amino acid residues was obtained by NH2-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase HPLC. This sequence was identical to the predicted NH2-terminal sequence of IL-6-R as previously reported. Analysis of the eluted proteins from both IFN-gamma and anti-IFN-gamma-R columns by inhibition of solid phase RIA, ELISA, SDS-PAGE, and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. Our finding, together with the already known presence of urinary TNF binding proteins and a soluble IL-2-R both in plasma and in urine, indicates that release of soluble cytokine receptors into body fluids is a general phenomenon that occurs under normal physiological conditions.

Reconstitution of Tubular Myelin from Synthetic Lipids and Proteins Associated with Pig Pulmonary Surfactant

Abstract: To analyze the mechanism of formation of tubular myelin (TM), we reconstituted TM from synthetic lipids and two surfactant-associated proteins (SP15 and SP35). SP15 was extracted from lyophilized pig pulmonary surfactant with 5% Triton X-100 and purified by DEAE-cellulose, CM-cellulose, and affinity chromatography with a specific antibody. SP35 was extracted from the precipitate of the 5% Triton X-100 extraction with pH 10 borate buffer and purified by DEAE-cellulose column chromatography. Lipid-SP15 complex was formed by a detergent dialysis method using octylglucopyranoside, and to this complex were added various concentrations of SP35 at 37° C. Structures similar to TM were formed when lipid-SP15 complex containing dipalmitoylphosphatidylcholine:phosphatidylglycerol from egg lecithin (2:1) and SP15 (lipid/protein, 5:1) was incubated with SP35 at concentrations of 0.15 to 0.22 mg/ml in CaCl2-containing buffer. At higher concentrations of SP35, many six-sided lattices were formed; the addition of EDTA ab...

Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transforming growth factor beta 2 affinity purification.

Abstract: Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.

Phosphotyrosyl protein phosphatases.

Abstract: Enzyme-catalysed reversible protein phosphorylation is an important cellular regulatory mechanism (Nimmo & Cohen, 1977; Krebs & Beavo, 1979). Regulatory protein phosphorylation occurs most frequently on seryl and threonyl residues (Taborsky, 1974), and less frequently, on tyrosyl residues (Hunter, 1982). Protein phosphotyrosine [Tyr(P)] normally accounts for only 0.01-0.050o of the total protein phosphoamino acid content of a normal cell (Hunter & Sefton, 1980), but this value increases to 1-3 when the cells are infected with viruses (Sefton et al., 1981; Martensen, 1982). In the last several years much attention has been focused on the phosphotyrosyl phosphorylation of cellular proteins by specific phosphotyrosyl kinases, and phosphotyrosyl phosphorylations have been associated with the regulation of cellular activities, including proliferation, differentiation, and transformation (Hunter & Sefton, 1982; Heldin & Westermark, 1984; Sefton & Hunter, 1984; Swarup et al., 1984; Sefton, 1985; Coughlin et al., 1988). The suggested association between phosphotyrosyl phosphorylation and cell proliferation was further supported by studies which showed that a number of polypeptide growth factor receptors [i.e. epidermal growth factor (EGF), platelet-derived growth factor, insulin-like growth factor-i, insulin, etc.] possess intrinsic phosphotyrosyl kinase activities that are activated when the growth factors are bound, and that the activation of the receptor phosphotyrosyl kinases is essential for the action of the growth factors (Cohen et al. 1980; Ek et al., 1982; Jacobs et al., 1983; Reynolds et al., 1981; Sefton & Hunter, 1984; Gammeltoft & Van Obberghen, 1986). Moreover, many oncogene products also act as phosphotyrosyl kinases, and share extensive sequence homology with several polypeptide growth factor receptors (Heldin & Westermark, 1984; Sefton, 1985; Pimental, 1987). Studies using site-directed mutagenesis (Chou et al., 1987; Kmiecik & Shalloway, 1987) and specific anti-(receptor phosphotyrosyl kinase) antibodies (Morgan et al., 1986; Morgan & Roth, 1987) have indicated that the phosphotyrosyl kinase activity of growth factor receptors is an essential component for the biological action of the growth factors and of the transforming proteins (Kmiecik & Shalloway, 1987). Together, these observations have led to the general conclusion that increased cellular phosphorylation of protein tyrosyl residues plays an important role in the action of growth factors and also serve to regulate the cellular growth processes in general (Hunter & Cooper, 1983). In order for phosphotyrosyl phosphorylation system to serve an important physiological regulatory mechanism, the process must be reversible [i.e. a process for removing the phosphate (Pi) from the phosphotyrosyl residues must exist]. The enzyme activities responsible for dephosphorylating phosphotyrosyl proteins are known as phosphotyrosyl protein phosphatases (PTPP). If phosphotyrosyl phosphorylation represents a general physiological regulatory mechahnism, it is reasonable to expect that the overall phosphQtyrosyl phosphorylation levels in cells are regulated by the balance of the activities of both phosphotyrosyl kinases and PTPPs. Thus, to achieve a complete understanding of the significance of this reversible enzymic covalent modification, it is necessary to know the nature and the regulation of, not only phosphotyrosyl kinases, but also PTPPs. The enzymology of phosphotyrosyl kinases has been intensively investigated (Brugge & Chinkers, 1983) and extensively reviewed (Sefton & Hunter, 1984; Hunter & Cooper, 1985; Sefton, 1985). However, relatively little is known of the nature of the PTPPs (Foulkes, 1983). Consequently, we will focus this review on the current status of our understanding of the nature and regulation of the PTPP activities. We will also summarize the recent evidence suggesting a physiological role for PTPP activity in the regulation of normal bone cell growth.

Isolation, partial amino acid sequence, and immunohistochemical localization of a brain-specific calcium-binding protein

Abstract: A calcium-binding protein (protein 10) having a molecular mass of 29 kDa and an isoelectric point of 5.3 was purified from guinea pig brain. The amino acid sequence of fragments from proteolytic digestion of protein 10 revealed an 86% sequence identity with a calcium-binding protein (calretinin) found in chicken retina. Polyclonal antibodies against protein 10 revealed a specific distribution of this protein within sensory neurons of auditory, visual, olfactory, nociceptive, and gustatory systems as well as other discrete neuronal circuits in rat and guinea pig brain, whereas no specific label was observed in any of several peripheral tissues examined.

Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein

Abstract: Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67,61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained (3H)leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 6 1 - and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. We propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a trans- membrane link between the extracellular matrix and the intracellular compartment.

Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides.

Abstract: Immunoglobulin heavy chain binding protein (BiP/GRP78) is a resident endoplasmic reticulum protein that binds tightly to a number of incompletely assembled or aberrant proteins BiP also binds ATP and can be purified by ATP affinity chromatography Here we show that an ATPase activity co-purifies with BiP prepared from canine pancreas The BiP-associated ATPase has a high affinity for ATP but a low turnover number, suggesting a regulatory, rather than an enzymatic role We also show that submicromolar levels of ATP or ADP decrease the rate of adsorption of [125I]BiP to nitrocellulose filters coated with protein or non-ionic detergents In contrast, micromolar levels of AMP increase the rate of adsorption Furthermore, ATP and ADP decrease the susceptibility of BiP to proteolytic degradation, whereas AMP was found to enhance degradation slightly Adenine nucleotides may therefore induce or stabilize different conformations of BiP even when ATP hydrolysis does not occur

Purification and characterization of human liver dehydroepiandrosterone sulphotransferase

Abstract: A form of sulphotransferase capable of sulphating dehydroepiandrosterone and other steroids was purified from cytosol prepared from human liver. Dehydroepiandrosterone sulphotransferase was purified 621-fold when compared with the activity in cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-bisphosphate-agarose affinity chromatography. During affinity chromatography, dehydroepiandrosterone sulphation activity could be resolved from p-nitrophenol sulphation activity catalysed by phenol sulphotransferase by using a gradient of adenosine 3'-phosphate 5'-phosphosulphate. The purified enzyme was most active towards dehydroepiandrosterone but was capable of conjugating a number of other steroids, including pregnenolone, androsterone and beta-oestradiol. No activity towards p-nitrophenol or dopamine, substrates for the phenol sulphotransferase, was observed with the pure enzyme. A single band with a subunit molecular mass of 35 kDa was observed by Coomassie Blue staining following SDS/polyacrylamide-gel electrophoresis of the purified enzyme. A molecular mass of 68-70 kDa was calculated for the active form of the enzyme by chromatography on Sephacryl S-200, suggesting that the active form of the enzyme is a dimer.

Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography.

Abstract: We recently found that many RNA polymerase II transcription factors are modified with N-acetylglucosamine residues These sugar moieties confer upon transcription factors an ability to bind the lectin wheat germ agglutinin We have taken advantage of this interaction to devise a purification procedure for the "GC-box" binding transcription factor Sp1 Crude nuclear extracts are first subjected to wheat germ agglutinin affinity chromatography and then subjected to sequence-specific DNA affinity chromatography The Sp1 protein purified by this procedure is at least 95% pure, and the overall recovery is greater than 80% In addition to yielding larger quantities of Sp1 than conventional schemes, the new purification procedure is also simpler and more rapid We show that wheat germ agglutinin affinity chromatography can also be used to purify the glycosylated forms of the CCAAT-binding transcription factor Thus, wheat germ agglutinin affinity chromatography may aid the purification of other transcription factors that bear N-acetylglucosamine residues Furthermore, the ability to separate glycosylated forms of transcription factors from their unglycosylated counterparts by wheat germ agglutinin affinity chromatography should facilitate investigations into the role of N-acetylglucosamine residues in the functioning of transcription factor proteins

Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography

Abstract: By using F-actin affinity chromatography columns to select proteins solely by their ability to bind to actin filaments, we have identified and partially purified greater than 40 proteins from early Drosophila embryos. These proteins represent approximately 0.5% of the total protein present in soluble cell extracts, and 2 mg are obtained by chromatography of an extract from 10 g of embryos. As judged by immunofluorescence of fixed embryos, 90% of the proteins that we have detected in F-actin column eluates are actin-associated in vivo (12 of 13 proteins tested). The distributions of antigens observed suggest that groups of these proteins cooperate in generating unique actin structures at different places in the cell. These structures change as cells progress through the cell cycle and as they undergo the specializations that accompany development. The variety of different spatial localizations that we have observed in a small subset of the total actin-binding proteins suggests that the actin cytoskeleton is a very complex network of interacting proteins.

Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics.

Abstract: Autoantibodies to phospholipid antigens can be characterized using solid phase immunoassays to detect anticardiolipin antibodies (ACA) or in phospholipid-dependent clotting tests where lupus anticoagulant (LA) activity can be demonstrated. It has not been established whether each activity is due to the same or separate antibody subgroups. Plasma from two patients with high levels of both activities were used for purification of ACA and LA using sequential ion-exchange, gel-filtration, and anti-Ig affinity chromatography. Plasma could be separated into fractions containing each activity in the absence of the other. In these fractions, antibodies responsible for LA activity do not bind to isolated phospholipids in solid phase immunoassays, and conversely antibodies binding in these assays (ACA) do not possess LA activity, suggesting LA are directed against a more complex lipid epitope. In addition, in one patient ACA was of IgG isotype only, whilst LA was due to IgG and IgM isotypes. In this patient, the IgG-ACA was heterogeneous with three peaks clearly separated on ion-exchange chromatography. Affinity purified antiphospholipid antibodies have been previously prepared from a number of patients using a phosphatidyl-serine column and antibodies purified in this manner possess ACA but not LA activity. Taken together, these data indicate that tests for ACA and LA define separable subgroups of phospholipid binding antibodies, thus explaining the discordance often seen between the two activities.

Localization of the receptor site for alpha-scorpion toxins by antibody mapping: implications for sodium channel topology.

Abstract: Site-directed and monoclonal antibodies recognizing different extracellular regions of the RII sodium channel alpha subunit have been used to determine the sequences that comprise the receptor for alpha-scorpion toxins by evaluating the effect of antibody on voltage-dependent binding of radio-labeled toxin isolated from Leiurus quinquestriatus to both reconstituted rat brain sodium channel and rat brain synaptosomes. Of six antibodies tested, two recognizing amino acid residues 355-371 and 382-400 located on an extracellular loop between transmembrane segments S5 and S6 of domain I and one recognizing residues 1686-1703 of a similar loop of domain IV inhibit binding by 30-55%. Inhibition is concentration-(EC50 = 0.4-2 microM) and time- (t1/2 = 40-80 min) dependent. Five different monoclonal antibodies recognizing the same extracellular loop in domain I inhibit binding completely with similar EC50 values as observed for site-directed antibodies. Kinetic studies of the antibody effect are consistent with a slowly reversible competition for the toxin receptor site. Our results suggest that the extracellular loops between segments S5 and S6 of domains I and IV comprise at least part of the alpha-scorpion toxin receptor site and support the membrane topology models in which domains I and IV are adjacent in the tertiary structure of the channel protein and six transmembrane sequences are contained in each of the four homologous domains.

Isolation of the human plasma corticotrophin-releasing factor-binding protein

Abstract: We report the purification of human corticotrophin-releasing factor-binding protein (hCRF-BP) using repeated affinity chromatography (with the aid of synthetic CRF immobilized on Sepharose 4B solid phase) followed by gel filtration. Presence of the binding protein was tracked throughout the procedure by its ability to inhibit binding of 125I-labelled hCRF to an hCRF antiserum; normal human plasma exhibits 80% inhibition in this system whereas sheep plasma, which does not contain an hCRF-BP, has no effect. Affinity cross-linking of 125I-labelled hCRF to the purified hCRF-BP was performed using disuccinimidyl suberate (1 mmol/l). SDS electrophoresis of the purified CRF cross-linked binding protein followed by radioautography resulted in one major band of Mr 37,000 which corresponded to our original molecular weight estimate based on the elution position of the binding protein on Sephacryl S-200. A 10(7)-fold purification of the hCRF-BP resulted in a preparation estimated to be 95% pure, with an overall yield of 5%.

Soluble lactose-binding vertebrate lectins: a growing family.

Abstract: Extracts of rat intestine contain nine soluble lactose-binding lectins with subunit molecular weights ranging from 14,500 to 19,000 that were purified by affinity chromatography and ion-exchange chromatography. Two of them are either identical with or closely related to other known rat lectins. A third appears to be the isolated carbohydrate-binding C-terminal domain of a known lectin but lacks the N-terminal domain presumed to mediate a different function. The others have not been described previously. Among them, the major rat intestinal lectin, RI-H, and a related protein, RI-G, have N-terminal amino acid sequences with similarities to sequences found in other known rat lectins. Therefore, these results introduce new members of a growing family of these structurally homologous soluble lactose-binding proteins.