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.
Vartanian
Amalia A
AA
Engelhardt Institute of Molecular Biology, Moscow, Russia. vartan@imb.ac.ru
D017209:Apoptosis; D002855:Chromatography, Thin Layer; D003599:Cytoskeleton / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D018260:Gelsolin / Q000502:physiology*; D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D017395:Plasminogen Activator Inhibitor 1 / Q000502:physiology*; D011485:Protein Binding; D012685:Sepharose / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013601:T-Lymphocytes / Q000378:metabolism
D015226:Dinucleoside Phosphates; D018260:Gelsolin; D017395:Plasminogen Activator Inhibitor 1; C011440:diadenosine triphosphate; D012685:Sepharose