Isolation of a 5-kilodalton actin-sequestering peptide from human blood platelets.

Abstract

Resting human platelets contain =0.3 mM unpolymerized actin. When freshly drawn and washed platelets are treated with saponin, 85-90% of the unpolymerized actin diffuses out. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions shows that the bulk of this unpolymerized actin migrates with a higher mobility than does pure G-actin, profllactin, or actin-kelsolin complex.When muscle G-actin is added to fresh or boiled saponin extract, the added muscle actin is shifted to the high-mobility form. The saponin extract contains an acidic peptide having a molecular mass in the range of 5 kDa, which has been purified to homogeneity by reverse-phase HPLC. This peptide also shifts muscle actin to the high-mobility form. Addition of either boiled saponin extract or the purified peptide to muscle G-actin also strongly and stoichiometrically inhibits salt-induced polymerization, as assayed by falling-ball viscometry and by sedimentation. We conclude that this peptide binds to the bulk of the unpolymerized actin in platelets and prevents it from polymerizing.It has been known for >10 years that many cell types contain a pool of unpolymerized actin (1, 2). Much of this actin can polymerize after purification (2, 3). Within the cell, polymerization is also induced by appropriate stimulation (1,4). The concentration of unpolymerized actin in cells can be up to 2 orders ofmagnitude greater than the critical concentration for assembly: in platelets, for example, the concentration of unpolymerized actin is estimated at 300-350 uM (5, 6), whereas the critical concentration is about 1 ,uM for the pointed end and about 0.2 uM for the barbed end (7). Furthermore, since the ion concentrations inside most cells favor actin polymerization, it seems inescapable that some other factor must be present to prevent this actin from polymerizing (8).The discovery that actin could form a 1:1 complex with either deoxyribonuclease I (9) or profilin (10) showed that actin monomers could be sequestered by other proteins and thereby prevented from polymerizing. Both profilin and profilactin have been isolated from blood platelets (11, 12); however, the role of profilin in maintaining the pool of unpolymerized actin in platelets has been clouded by several discrepancies. First, early estimates ofthe quantity ofprofilin in platelets suggested that nearly all of the unpolymerized actin in platelets could be sequestered as profilactin (11,12), but subsequently Lind et al. (13) used the polyproline affinity technique and estimated a profilin concentration that is well below the concentration of unpolymerized actin. Second, the binding constant of profilin for actin appears to be relatively weak, in the range of 1-6 ILM (7,14). Thus, even ifthe profilin concentration were equal to the concentration of unpolymerized actin in platelets, the concentration offree G-actin would be in the range of 20 kLM, which is at least an order of magnitude above the critical concentration for the pointed end. Third, Lind et al. (13) have also r...

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

“…Therefore, the high intracellular pool of monomeric or (G-)actin can only maintained by binding to specific AbPs, which by formation of complexes inhibit G-actin polymerization. Thus the intracellular pool of monomeric actin can be divided in two fractions: a small amount of free G-actin that is in rapid equilibrium with filamentous actin and a larger pool of sequestered monomeric actin, which by complexation to AbPs like profilin or b-thymosins is inhibited in its ability to polymerize and therefore removed from the cycling actin pool [Safer et al, 1990[Safer et al, , 1991 for review see also Huff et al, 2001].…”

Vertebrate β‐thymosins: Conserved synteny reveals the relationship between those of bony fish and of land vertebrates

“…First identified from a cytokine-like activity [2], the archetypical b-thymosin, b4 was later found to be an intracellular protein that sequesters G-actin [3] and thereby influences microfilament dynamics.…”

Relationships between Actin Regulatory Mechanisms and Measurable State Variables

“…23,48 Measurements of the concentration of profilin in cells range from 5 lM in chick brain 12 to 55 lM in platelets. 17 However, not all profilin is necessarily available for binding to actin, as profilin has over 50 identified binding partners, 58 and in particular, it has been demonstrated that binding to phosphoinositides dissociates profiling-actin complexes.…”

“…28 While not interacting with filaments directly, thymosins bind actin monomers to help maintain roughly equal amounts of polymerized and unpolymerized actin in cells. 48 To visualize the dynamics of actin in cells, investigators follow the motions of fluorescent actin tracers introduced by microinjection or ectopic expression. Traditionally, investigators have used fluorescence recovery after photobleaching (FRAP) or photoactivation of fluorescence (PAF) experiments to locally perturb fluorescence.…”