Thymosin β4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes

Abstract

Thymosin β4 (Tβ4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that Tβ4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain ∼169 fg/cell ± 90 fg/cell (SD), corresponding to a cytoplasmic concentration of ∼49 ± 80.5 μM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/Tβ4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/Tβ4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of Tβ4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenylactin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with Tβ4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen Tβ4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.