Showing papers on "Affinity chromatography published in 1993"
TL;DR: Small peptide substrates that are enzymatically biotinylated at a single site should be useful for a variety of purposes in labeling, purification, detection, and immobilization of proteins.
Abstract: I describe a technique for screening peptide libraries of over 10(9) independent clones for substrates of peptide-modifying enzymes. The peptides, linked to their genetic material by the lac repressor, are exposed to the enzyme and then screened by affinity purification on a receptor specific for the modified product. The enzyme characterized, E. coli biotin holoenzyme synthetase, normally adds biotin to a specific lysine residue in complex protein domains. The 13 residue substrate identified by this library screening approach is much smaller than the 75 amino acid required sequence of the natural substrate, and can function at either end of a fusion protein. The sequence is quite distinct at some positions from that region of the natural substrates, presumably because the peptides have to mimic the folded structure formed by the natural substrate. This technique should be useful for mapping the substrate specificity of a variety of peptide-modifying enzymes. In addition, small peptide substrates that are enzymatically biotinylated at a single site should be useful for a variety of purposes in labeling, purification, detection, and immobilization of proteins.
641 citations
TL;DR: The use of a novel kind of random peptide library for the stepwise engineering of a C-terminal fusion peptide which confers binding activity towards streptavidin is described in this study.
Abstract: The facile detection and purification of a recombinant protein without detailed knowledge about its individual biochemical properties constitutes a problem of general interest in protein engineering. The use of a novel kind of random peptide library for the stepwise engineering of a C-terminal fusion peptide which confers binding activity towards streptavidin is described in this study. Because of its widespread use as part of a variety of conjugates and other affinity reagents, streptavidin constitutes the binding partner of choice both for detection and purification purposes. The streptavidin-affinity tag was engineered at the C-terminus of the VH domain as part of the D1.3 Fv fragment which was functionally expressed in Escherichia coli. Irrespective of whether it was displayed by the VH or the VL domain, the optimized version of the affinity peptide termed 'Strep-tag' allowed the detection of the Fv fragment both on Western blots and in ELISAs by a streptavidin-alkaline phosphatase conjugate. In addition, the one-step purification of the intact Fv fragment carrying a single Strep-tag at the C-terminus of only one of its domains was achieved by affinity chromatography with streptavidin-agarose using very mild elution conditions.
325 citations
TL;DR: Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp 90, which appears to comprise an important functional unit in the assembly of progesterone receptor complexes.
Abstract: Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.
275 citations
TL;DR: The advantages and restrictions of tagging recombinant proteins with histidines and purifying them by Ni2+-NTA chromatography are discussed.
Abstract: Expression of recombinant proteins is a standard technique in molecular biology and a wide variety of prokaryotic as well as eukaryotic expression systems are currently in use. A limiting step is often the purification of the expressed recombinant protein, particularly if mammalian expression systems that yield low expression levels are employed. Here, we discuss the advantages and restrictions of tagging recombinant proteins with histidines and purifying them by Ni(2+)-NTA chromatography.
273 citations
TL;DR: Data show that protein S has an anticoagulant function which is independent of activated protein C and, at least in part, that this is because of its competition with prothrombin for direct binding to Factor Va.
209 citations
TL;DR: It is suggested that the GluR1 subunit consists of at least two isoforms possessing a common N-terminal region but a distinct C-terminus, and that the hippocampus, septum, amygdala and olfactory bulb exhibited the strongest immunoreactivity.
197 citations
TL;DR: In conclusion, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins.
Abstract: The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC). To identify components which interact physically with this yeast nucleoporin, the tagged C-terminal domain of NSP1 (ProtA-NSP1) was isolated by affinity chromatography under non-denaturing conditions. The purified complex contains ProtA-NSP1, two previously identified 'GLFG' nucleoporins, NUP49 (NSP49) and p54 and a novel protein designated NIC96 (for Nucleoporin-Interacting Component of 96 kDa). Conversely, affinity purification of tagged NSP49 enriches for NSP1, the p54 and the NIC96 component. The NIC96 gene was cloned; it encodes a novel 839 amino acid protein essential for cell growth. By immunofluorescence, protein A-tagged NIC96 exhibits a punctate nuclear membrane staining indicative of nuclear pore location. Therefore, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins.
189 citations
TL;DR: Homology analyses confirm that ERBC is a new member of the cyclophilin family and contains a C-terminal domain with significant sequence homology to an internal region of p59, a binding protein for the immunosuppressant FK506 (FKBP59).
188 citations
TL;DR: A novel method of purifying inclusion bodies, using the detergent octyl-polyoxyethylene (octyl-POE), as well as establishing methods for preventing fusion protein proteolytic breakdown are developed, which had the same amino acid content, aa sequence, gel electrophoretic mobility and antibacterial activity as CEME produced by protein chemical procedures.
181 citations
TL;DR: The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity and the enzymatic activity of the purified material was shown to be a general NTP enzyme and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides.
Abstract: The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses. Images
178 citations
TL;DR: When receptor populations were immunoprecipitated by the alpha 5-subunit antiserum from different brain regions, zolpidem displayed striking differences in affinity pointing to the role of subunits other than alpha 5 in determining receptor affinity.
TL;DR: Antisera raised against the plant glycoproteins β-fructosidase and horseradish peroxidase can be fractionated on an affinity column of honeybee venom phospholipase A2 to produce serum fractions that are specific for either the α1 → 3 frucose or β1 → 2 xylose epitopes commonly found on the Asn-linked glycans of plant glyCoproteins.
TL;DR: The single-gene construct approach provides a way of generating an immunoglobulin-like molecule which retains the specificity, binding properties, and cytolytic activity of the chimeric mAb CC49, potentially a therapeutic and diagnostic reagent against a range of human carcinomas.
Abstract: We describe construction of a single gene encoding a single-chain immunoglobulin-like molecule. This single-gene approach circumvents inefficiencies inherent in delivering two genes into a mammalian cell and in the assembly of a functional immunoglobulin molecule. It would also facilitate ex vivo transfection of cells for gene-therapy protocols. SP2/0 murine myeloma cells transfected with the single gene SG delta CLCH1 expressed a single-chain protein, SC delta CLCH1, comprising approximately 60 kDa of the anti-carcinoma monoclonal antibody (mAb) CC49. The single-chain protein consisted of the heavy- and light-chain variable (VH and VL) domains of the mAb covalently joined through a short linker peptide, while the carboxyl end of the VL domain was linked to the amino terminus of the human gamma 1 Fc region through the hinge region. The single-chain protein assembled into a dimeric molecule, termed SCA delta CLCH1, of approximately 120 kDa and was secreted into the tissue culture fluid. SDS/PAGE analysis of the secreted immunoglobulin purified by protein G affinity chromatography confirmed the size of the molecule. The native mAb CC49 and SCA delta CLCH1 of CC49 showed similar binding to the tumor-associated glycoprotein TAG-72, and the chimeric mAb CC49 and SCA delta CLCH1 showed similar cytotoxic activity. This single-gene construct approach provides a way of generating an immunoglobulin-like molecule which retains the specificity, binding properties, and cytolytic activity of the chimeric mAb CC49. The immunoglobulin-like molecule SCA delta CLCH1 is potentially a therapeutic and diagnostic reagent against a range of human carcinomas.
TL;DR: The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar, were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products.
Abstract: The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S., Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207–212], were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An α-agarase, i.e specific for the α(13) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatograpy (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified α-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0–9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45°C or by removing calcium. In addition, a β-galactosidase specific for the end products of the α-agarase was present in the α-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.
TL;DR: In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.
Abstract: A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized. Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal. Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins. Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols. Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography. Purification was independent of recombinant protein solubility in sonicated or freeze-thawed E. coli lysates. Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitation, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.
TL;DR: The SRS2 protein was found to have in vitro DNA-dependent ATPase and DNA helicase activities and the carboxyl-terminal region of the protein is shown to contain a sequence for nuclear localization.
Book•
01 Jan 1993
TL;DR: Determination of Cell Surface Polarity by Solid-Phase Lactoperoxidase Iodination and Determination of the Transverse Topography of Membrane Lipids Using Enzymes and Covalent Labels as Probes.
Abstract: Crystallization of Membrane Proteins for X-Ray Analysis. Determination of Cell Surface Polarity by Solid-Phase Lactoperoxidase Iodination. Biochemical Methods to Determine Cell Surface Topography: Part A: Labeling of Oxidized Glycoproteins with 3H-Borohydride Part B: Identification and Separation of Integral Membrane Proteins Using Triton X-114 Part C: Surface-Selective Labeling of Polarized Proteins on Tight Epithelia Using Sulfo-N-Hydroxysuccinimido-Biotin (S-NHS-B). Use of Antipeptide Antibodies for the Isolation and Study of Membrane Proteins: Part A: Preparation of Antibodies Part B: Affinity-Purification of Antipeptide Antibodies Part C: Purification of Membrane Proteins by Immunoaffinity Chromatography Part D: Competitive ELISA for Determining Membrane Protein Topology. The Production of Monoclonal Antibodies to Membrane Proteins: Part A: Production of Hybridoma Cell Lines Part B: Screening for Monoclonal Antibody Production. Purification of a Membrane Protein (Ca2+/Mg2+-ATPase) and Its Reconstitution into Lipid Vesicles. Measurement of Protein-Protein Interactions in Reconstituted Membrane Vesicles Using Fluorescence Spectroscopy. Measurement of Lipid-Protein Interactions in Reconstituted Membrane Vesicles Using Fluorescence Spectroscopy. Determination of the Transverse Topography of Membrane Lipids Using Enzymes and Covalent Labels as Probes. Determination of the Transverse Topography of Membrane Phospholipids Using Phospholipid Transfer Proteins as Tools. Prothrombinase Complex as a Tool to Assess Changes in Membrane Phospholipid Asymmetry. Fluorescent Glycerolipid Probes: Synthesis and Use for Examining Intracellular Lipid Trafficking: Part A: Synthesis of Fluorescent Glycerolipid Probes Part B: Delivery of Fluorescent Glycerophospholipid Probes to Cultured Cells. Synthesis and Use of Spin-Labeled Lipids for Studies of the Transmembrane Movement of Phospholipids. Measurement of Membrane Fluidity and Membrane Fusion with Fluorescent Probes Extraction and Assay of Cyclic Nucleotides. Analysis of G-Proteins Regulating Signal Transduction Pathways: Part A: Identification of G-Proteins Part B: GTPase Studies Part C: Reconstitution of Second Messenger Pathways in Permeabilized Cell Preparations Using GTP Analogs and Receptor Ligands. Assay of Protein Kinases and Protein Phosphorylation: Part A: Protein Kinase-Mediated Phosphorylation Events Part B: Measurement of Protein Kinase Expression Part C: Measurement of Protein Kinase Activity and Translocation. Analysis of Cellular Phosphoinositides and Phosphoinositols: Extraction and Simple Analytical Procedures: Part A: Biosynthesis and Extraction of Phosphoinositides and Phosphoinositols Part B: Separation of Phosphoinositols by Anion-Exchange Chromatography Part C: Separation of Phosphoinositides by Thin-Layer Chromatography Part D: Deacylation of Phospholipids and Separation of Products by Anion-Exchange Chromatography. Analysis of Cellular Phosphoinositides and Phosphoinositols by High Performance Liquid Chromatography: Part A: Sample and Standards Preparation Part B: HPLC Analytical Methods. Cytosolic Free Calcium Measurements in Single Cells Using Calcium-Sensitive Fluorochromes. Membrane Permeabilization with Bacterial Toxins. Measurement of Ion Fluxes and pH Gradients Across Cell Membranes. Ligand Binding and Processing: The Perfused Liver as a Model System. The Binding of Protein-Ligands to Cell-Surface Receptors. Appendix: 1. Density Gradient Media. Appendix: 2. Balanced Salt Solutions. Index.
TL;DR: Heterogeneous sized pools and affinity-fractionated subpools of heparin-derived oligosaccharides were quantitatively assessed as inhibitors or enhancers of specific bFGF-mediated biological activities and inhibited the interaction between cell surface heparan sulfate proteoglycan and bF GF.
TL;DR: Comparison of the chemical and immunological properties of rat liver m-AspAT with those ofRat liver FABPpm isolated by two procedures revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, and isoelectric point.
Abstract: A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.
TL;DR: A 64-kDa protein, p64, identified as a component of this chloride channel was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity and has now been used to identify a clone, H2B, from a kidney cDNA library.
TL;DR: It is suggested that this glycoprotein is not a “classical” AGP, but represents the first example of a new class of hydroxyproline-poor AGPs.
Abstract: Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.
Journal Article•
TL;DR: The conditioned medium of the murine macrophage PU5.1.8 was analyzed by two-dimensional gel electrophoresis in order to detect LPS-induced proteins, and several monokines (TNF-alpha and macrophages inflammatory protein-1 alpha and 1 beta) were identified as L PS-induced spots.
Abstract: The conditioned medium of the murine macrophage PU5.1.8 was analyzed by two-dimensional gel electrophoresis in order to detect LPS-induced proteins. Spots of interest were identified by microsequencing of internal peptides generated by limited in situ acid hydrolysis. In total conditioned medium, several monokines (TNF-alpha and macrophage inflammatory protein-1 alpha and 1 beta) were identified as LPS-induced spots. Because minor spots could be masked by the complexity of the 2-D pattern, conditioned medium was successively fractionated by zinc precipitation and affinity chromatography (Procion red and Con A agarose). Zinc supernatant fraction, Procion red flow-through, and Con A eluate fractions were further analyzed by 2-D gel electrophoresis for the presence of LPS-induced spots. In these fractions serum amyloid A3, lipocalin 24p3, cathepsin B, and plasminogen activator inhibitor-I were characterized as LPS-induced proteins secreted by macrophages. Lipocalin 24p3 protein was retrieved for the first time. In addition to these proteins that follow a classical secretory pathway, several cellular proteins (mainly ribosomal proteins) were retrieved as LPS-induced proteins in the conditioned medium. Control experiments argue against the obvious explanation that the latter observation is caused solely by cellular leakage.
TL;DR: Findings indicate that the plasma membrane of CHO cells contains approximately half of the total cellular phospholipids and an even higher percentage of sphingomyelin and cholesterol.
TL;DR: The sequence information for the isolated GST, which is designated “GST IV” indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.
Abstract: A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.
TL;DR: It is concluded that human LTC4 synthase is a glutathione S-transferase composed of an 18-kDa polypeptide that is enzymatically active as a homodimer and may be phosphoregulated in vivo.
Abstract: Human leukotriene C4 (LTC4) synthase was purified > 25,000-fold to homogeneity from the monocytic leukemia cell line THP-1. Beginning with taurocholate-solubilized microsomal membranes, LTC4 synthase was chromatographically resolved by (i) anion exchange, (ii) affinity chromatography (through a resin of biotinylated LTC2 immobilized on streptavidin-agarose), and then (iii) gel filtration. The final preparation contained only an 18-kDa polypeptide. The molecular mass of the pure polypeptide was consistent with an 18-kDa polypeptide from THP-1 cell membranes that was specifically photolabeled by an LTC4 photoaffinity probe, 125I-labeled azido-LTC4. On calibrated gel-filtration columns, purified LTC4 synthase activity eluted at a volume corresponding to 39.2 +/- 3.3 kDa (n = 12). The sequence of the N-terminal 35 amino acids was determined and found to be a unique sequence composed predominantly of hydrophobic amino acids and containing a consensus sequence for protein kinase C phosphorylation. We therefore conclude that human LTC4 synthase is a glutathione S-transferase composed of an 18-kDa polypeptide that is enzymatically active as a homodimer and may be phosphoregulated in vivo.
TL;DR: A role for the 5A11 antigen as a novel recognition molecule in the developing neural retina is established after microsequencing of the major component isolated by immunoaffinity chromatography identified the HT7 antigen, a unique member of the immunoglobulin super gene family (IGSF), as a homologous if not identical protein to the 5 A11 antigen.
Abstract: In continuing efforts to identify cell-surface molecules involved in cell-cell interactions in the developing avian retina, we identified a monoclonal antibody, the 5A11 antibody, which possessed the ability to interfere with contact-dependent glial cell maturation in vitro. We sought to determine the molecular and biochemical identity of the glycoprotein recognized by this antibody, and using additional criteria, establish whether the 5A11 antigen is indeed a cell-recognition molecule in the developing retina. Immunohistochemical analyses demonstrate that in the hatchling chick retina and in live cultures of embryonic retina cells, the 5A11 antigen is predominantly associated with Muller glial cells whereas little is observed on neuronal elements. Microsequencing of the major component isolated by immunoaffinity chromatography identifies the HT7 antigen (Seulberger et al.: EMBO Journal 9:2151-2158, 1990), a unique member of the immunoglobulin super gene family (IGSF), as a homologous if not identical protein to the 5A11 antigen. The HT7 antibody, furthermore, recognizes affinity purified 5A11 antigen, and both the HT7 antibody and additional probes generated against the 5A11 antigen recognize a major polypeptide of 45.5 kDa and a minor band of 69 kDa on Western blots of membrane preparations from neural retina. To verify that the 5A11 antigen mediates cell-cell recognition events in the developing neural retina, we examined the consequences of adding antibody to monolayer cultures of dissociated embryonic retina cells and to dissociated retina cells in rotation-mediated suspension culture. Addition of the 5A11 antibody to monolayer cultures results in alteration in the development of the stereotypic arrangement of neurons and glia characterized by a reduction in the number and complexity of neural extensions upon the glial-derived flat cells. Similarly, addition of antibodies generated against the 5A11 antigen to dissociated cells in rotation cultures significantly reduces retina cell reaggregation as monitored by computer-assisted image analysis of cell aggregate size. These data and the identification of the 5A11 antigen as a member of the IGSF establish a role for the 5A11 antigen as a novel recognition molecule in the developing neural retina.
TL;DR: Data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.
Abstract: We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.
TL;DR: A plasmid for direct expression in Escherichia coli of the mature form bovine adrenodoxin reductase was constructed from the full-size cDNA for the enzyme and an expression vector pCWori+.
Abstract: A plasmid for direct expression in Escherichia coli of the mature form bovine adrenodoxin reductase was constructed from the full-size cDNA for the enzyme [Y. Sagara, Y. Takata, T. Miyata, T. Hara, and T. Horiuchi, J. Biochem. (Tokyo), 102, 1333 (1987)] and an expression vector pCWori+. The recombinant adrenodoxin reductase was purified from the transformed E. coli cell lysates using adrenodoxin-Sepharose affinity chromatography [T. Sugiyama and T. Yamano, FEBS Lett., 52, 145 (1975)] with a yield of 2.5 mg/l of culture. The purified recombinant enzyme showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and migration was identical with that of the authentic enzyme purified from bovine adrenal cortex mitochondria. The recombinant enzyme had Ser at its amino-terminus and the sequence of the amino terminal 9 residues was identical with that of the authentic bovine enzyme. The absorption spectrum of the recombinant enzyme showed peaks at 270, 376, and 450 nm and shoulders at 425 and 475 nm. Flavin content of the recombinant enzyme was 0.8 mol FAD/mol. The apparent Km value for bovine adrenodoxin in NADPH-cytochrome c reductase activity using a reconstitution system was 16 nM, a value comparable with that of the authentic bovine enzyme (17 nM). The cholesterol side chain cleavage activity with a reconstitution system was about 75% of that obtained when the authentic enzyme was used.
TL;DR: It is demonstrated that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in aprotein L-based purification protocol.
TL;DR: It is concluded that interference from carbamylated hemoglobin explains only a small part of existing discrepancies between results of glycohemoglobin assays in current use.
Abstract: In vitro-synthesized carbamylated and acetylated hemoglobins interfered in assays of glycohemoglobin by HPLC and electrophoresis but had no effects on results obtained by affinity chromatography and enzyme immunoassay. Correlations between long-term serum urea concentrations and glycohemoglobin percentages revealed that, in vivo, carbamylated hemoglobin equivalent to 0.063% of total hemoglobin is formed for every 1 mmol/L of serum urea. The use of acetylsalicylate, either chronically in small doses (200-300 mg/day) or for 1 week at 2000 mg/day, did not cause significant interference from acetylhemoglobin, formed in vivo. We conclude that interference from carbamylated hemoglobin explains only a small part of existing discrepancies between results of glycohemoglobin assays in current use. The interfering effect of acetylhemoglobin formed in vivo with acetyl-CoA as substrate is as yet unknown.