Affinity chromatography: a review of clinical applications.
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Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with other analytical methods, such as reversed-phase liquid chromatography, gas chromatography, and capillary electrophoresis. Indirect analyte detection methods are also described in which immunoaffinity chromatography is used to perform flow-based immunoassays. Other applications that are reviewed include affinity-based chiral separations and the use of affinity chromatography for the study of drug or hormone interactions with binding proteins. Some areas of possible future developments are then considered, such as tandem affinity methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or aptamers as affinity ligands for clinical analytes. (+info)
Enhancing activity and phospholipase A2 activity: two independent activities present in the enhancing factor molecule.
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Enhancing factor (EF), a molecule that increases the binding of epidermal growth factor (EGF) to A431 cells, was first isolated in our laboratory from mouse intestines, and subsequently shown to be a secretory form of phospholipase A2 (PLA2) [Mulherkar, Rao, Wagle, Patki and Deo (1993) Biochem. Biophys. Res. Commun. 195, 1254-1263]. We had proposed earlier that EF increases the binding of EGF by first binding to its own cell-surface receptor [identified as a 100 kDa molecule; Mulherkar and Deo (1986) J. Cell. Physiol. 127, 183-188], and then by creating a binding site for EGF. However, due to its PLA2 activity, there was a possibility that EF, by its phospholipase activity could be unmasking cryptic EGF receptors on the cell surface, thereby increasing the number of binding sites for EGF. To test whether enhancing activity and phospholipase activity are independent of each other, a series of mutations were created using the full-length EF cDNA as a template, expressed in 293 cells and the mutant recombinant proteins checked for EF as well as PLA2 activities. Our studies have shown that one of the mutant EF proteins, lacking PLA2 activity, retains EF activity. This demonstrates unambiguously that EF and PLA2 activities are two independent activities in the same molecule. Mutation in the Ca2+-binding loop resulted in loss of EF activity, thereby demonstrating that EF activity is Ca2+-dependent. The N-terminal region of the EF molecule appears to be crucial for the enhancing activity. (+info)
Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.
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Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd+info)
Regulation of alpha-helical coiled-coil dimerization in chicken skeletal muscle light meromyosin.
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The dimerization specificity of the light meromyosin (LMM) domain of chicken neonatal and adult myosin isoforms was analyzed by metal chelation chromatography. Our results show that neonatal and adult LMMs associate preferentially, although not exclusively, as homodimeric coiled-coils. Using chimeric LMM constructs combining neonatal and adult sequences, we observed that a stretch of 183 amino acids of sequence identity at the N terminus of the LMM was sufficient to allow the adult LMM to dimerize in a non-selective manner. In contrast, sequence identity in the remaining C-terminal 465 amino acids had only a modest effect on the dimerization selectivity of the adult isoform. Sequence identity at the N terminus also promoted dimerization of the neonatal LMM to a greater degree than sequence identity at the C terminus. However, the N terminus had only a partial effect on the dimerization specificity of the neonatal sequence, and residues distributed throughout the LMM were capable of affecting dimerization selectivity of this isoform. These results indicated that dimerization preference of the neonatal and adult isoforms was affected to a different extent by sequence identity at a given region of the LMM. (+info)
Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors.
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Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A2 (TXA2) receptor-Galphaq complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to Galphaq, purification of TXA2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G12 family. Using an antipeptide antibody specific for the human G13 alpha-subunit, this G-protein was identified as Galpha13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 alpha-subunit. Further evidence for functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that U46619 induced a significant decrease in Galphaq and Galpha13 association with the receptor protein. These results indicate that both Galphaq and Galpha13 are functionally coupled to TXA2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous Galpha13 is a TXA2 receptor-coupled G-protein, as: 1) its alpha-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPgammaS binding to Galpha13, and 3) Galpha13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation. (+info)
Escherichia coli DNA topoisomerase I copurifies with Tn5 transposase, and Tn5 transposase inhibits topoisomerase I.
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Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. Genetic evidence suggested that this killing involves titration of E. coli topoisomerase I (Topo I). Here, we present biochemical evidence that supports this model. Tn5 Tnp copurifies with Topo I while nonkilling derivatives of Tnp, Delta37Tnp and Delta55Tnp (Inhibitor [Inh]), show reduced affinity or no affinity, respectively, for Topo I. In agreement with these results, the presence of Tnp, but not Delta37 or Inh derivatives of Tnp, inhibits the DNA relaxation activity of Topo I in vivo as well as in vitro. Other proteins, including RNA polymerase, are also found to copurify with Tnp. For RNA polymerase, reduced copurification with Tnp is observed in extracts from a topA mutant strain, suggesting that RNA polymerase interacts with Topo I and not Tnp. (+info)
Purification, kinetic properties, and intracellular concentration of SpoIIE, an integral membrane protein that regulates sporulation in Bacillus subtilis.
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SpoIIE is a bifunctional protein which controls sigmaF activation and formation of the asymmetric septum in sporulating Bacillus subtilis. The spoIIE gene of B. subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated. (+info)
The nature of the subset of MHC class II molecules carrying the CDw78 epitopes.
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A CDw78 mAb FN1 was shown to recognize DP and/or DR molecules under the conditions of Western blotting. DP molecules were specifically retarded on a column of the FN1 immunosorbent; binding of FITC-labeled FN1 to B cell lines was completely blocked by excess of mAb to DR/DP beta chains, partially by several mAb to DP and weakly by some mAb to DR. The binding of two other CDw78 mAb, FN4 and MR11, to the B cell surface was most strongly inhibited by excess of different mAb to DR. Kinetics of stable binding of the CDw78 mAb indicated that their monovalent binding is of low affinity and that the stable binding to the surface is due to bivalent binding to two spatially close MHC class II molecules. FN1-based immunosorbent effectively immunoisolated complexes of MHC class II proteins with several tetraspanin molecules from a mild detergent lysate of a B cell line. It is concluded that FN1 and most likely also the other two CDw78 mAb recognize with low affinity determinants on MHC class II molecules (DP or DR) and preferentially bind in a stable fashion to dimerized or aggregated MHC class II molecules. Such dimers or aggregates may either exist as preformed on the cell surface or may be gradually formed and stabilized by bivalent interaction with mAb. These structures may be related to the previously described 'superdimers' of MHC class II and/or 'MHC-tetraspanin complexes'. CDw78 mAb may be valuable tools targeting such aggregated fraction of MHC class II molecules which can exhibit important signaling and antigen-presenting properties. (+info)