Covalent binding of 3-pyridinealdehyde nicotinamide adenine dinucleotide and substrate to glyceraldehyde 3-phosphate dehydrogenase.
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Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phoshate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12) forms a complex with 3-pyridinealdehyde-NAD which survives precipitation with 7% perchloric acid. The molar ratio bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD. 3-Pyridinealdehyde-deamino-NAD or glyceraldehyde 3-phosphate also forms an acid-stable complex with glyceraldehyde 3-phosphate dehydrogenase; however, NAD, 3-acetylpyridine-NAD, or thionicotinamide-NAD does not produce an acid-stable complex. Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by[14-C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphates activity of the enzyme for which the active site sulfhydryl residues must be oxidized. (+info)
A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix.
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Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality. (+info)
Iodination of salicylic acid improves its binding to transthyretin.
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Transthyretin (TTR) is a plasma homotetrameric protein associated with senile systemic amyloidosis and familial amyloidotic polyneuropathy. In theses cases, TTR dissociation and misfolding induces the formation of amyloidogenic intermediates that assemble into toxic oligomeric species and lead to the formation of fibrils present in amyloid deposits. The four TTR monomers associate around a central hydrophobic channel where two thyroxine molecules can bind simultaneously. In each thyroxine binding site there are three pairs of symmetry related halogen binding pockets which can accommodate the four iodine substituents of thyroxine. A number of structurally diverse small molecules that bind to the TTR channel increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis have been tested. In order to take advantage of the high propensity to interactions between iodine substituents and the TTR channel we have identified two iodinated derivatives of salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid, available commercially. We report in this paper the relative binding affinities of salicylic acid and the two iodinated derivatives and the crystal structure of TTR complexed with 3,5-diiodosalicylic acid, to elucidate the higher binding affinity of this compound towards TTR. (+info)
DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.
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On the selection of a tracer for PET imaging of HER2-expressing tumors: direct comparison of a 124I-labeled affibody molecule and trastuzumab in a murine xenograft model.
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The identification and purification of the heterotrimeric GTP-binding protein from squid (Loligo forbesi) photoreceptors.
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The principal GTP-binding protein (G-protein) from squid (Loligo forbesi) photoreceptor membranes has been identified by amino acid sequencing. The heterotrimeric protein was purified by detergent solubilization and ion-exchange chromatography. The amino acid sequence of the G-protein alpha-subunit (G-alpha) indicates that this subunit is closely related to the recently characterized Gq subgroup, whereas the G-gamma subunit varies widely in sequence from other homologues. (+info)
Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions.
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When the enzymatically active A-fragment of diphtheria toxin is translocated to the cytosol, the B-fragment inserts into the membrane in such a way that a 25-kDa polypeptide becomes shielded from proteases added to the external medium. We have attempted to determine the boundaries of this polypeptide within the toxin B-fragment as well as the topology of the B-fragment in the membrane. Chemical cleavage of the 25-kDa polypeptide with hydroxylamine and o-iodosobenzoic acid yielded fragments of sizes indicating that the 25-kDa polypeptide starts at residue approximately 300 and extends to the COOH-terminal end. Experiments where the toxin was labeled with [35S]cysteine at distinct positions of the B-fragment supported this conclusion. Treatment of cells with inserted B-fragment with L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin and with V8 protease from Staphylococcus aureus yielded protected 27- and 30-kDa fragments in addition to 25 kDa, indicating that the region 240-264 is also at the outside. The topology of the inserted B-fragment is discussed. (+info)