sepharose has been researched along with diadenosine-triphosphate* in 1 studies
1 other study(ies) available for sepharose and diadenosine-triphosphate
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Gelsolin and plasminogen activator inhibitor-1 are Ap3A-binding proteins.
Previous data on the accumulation of diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) in cells in response to various physiological factors raised the issue of identification of Ap3A binding proteins as potential targets for Ap3A. Ap3A binding proteins were isolated from a human leukocyte lysate by affinity chromatography through Ap3A-aga-rose. Two proteins, gelsolin and plasminogen activator inhibitor-1 (PAI-1), were tentatively identified by in-gel tryptic digestion and mass fingerprint analysis by MALDI-TOF mass spectrometry. The ability of the pure proteins to bind Ap3A was confirmed. Scatchard analysis of [3H]Ap3A binding data yielded dissociation constants of 0.3 microM for gelsolin and 4.1 microM for PAI-1. Binding was saturable at 0.78 mol Ap3A/mol of gelsolin and 0.68 mol Ap3A/mol PAI-1. The binding was non-covalent and insensitive to the presence of divalent metal ions. In neither case was binding affected by a 100-fold molar excess of ATP, ADP and AMP or Ap4A, suggesting a high degree of specificity for Ap3A. Ap3A produced significant effects on cell morphology when added at 10 microM to reversibly permeabilized CEM-SS cells, suggesting that it might influence cytoskeletal disruption by activating gelsolin. Ap3A added externally to HL60 promyelocytic cells reduced the inhibitory effect of PAI-1 on VP16-induced apoptosis. These findings provide new information about intra- and extracellular targets of Ap3A. Topics: Apoptosis; Chromatography, Thin Layer; Cytoskeleton; Dinucleoside Phosphates; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gelsolin; HL-60 Cells; Humans; Immunohistochemistry; Kinetics; Leukocytes; Microscopy, Phase-Contrast; Plasminogen Activator Inhibitor 1; Protein Binding; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; T-Lymphocytes | 2003 |