Actin containing structures in the cytoplasm of non-muscle cells have
been implicated in cell locomotion and control of the distribution of
cytoplasmic and cell surface components. Consistent with this versatility of
actin function is the complexity of actin containing structures that are
found in non-muscle cells.
Actin is a highly conserved protein with identical functional properties
from cell to cell. Recently a number of actin binding proteins have been
purified that may account for the assembly of actin into the different
structures found in vivo. We have been investigating the actin binding
proteins that are responsible for Ca++ regulated gelation of actin that was
first documented in cell free extracts of Dictyostelium discoideum, a
cellular slime mold. In this case these actin binding proteins will be
referred to as gelation factors. Our method for purifying the gelation
factors from Dictyostelium is briefly outlined as follows. Cell free
extracts that contained gelation activity were fractionated with ammonium
sulfate into O-45 and 45-60% pellets. Gelation activity in each pellet was
purified by chromatography. The 45-60% pellet contained 95% of the gelation
activity in the extract and was resolved into two gelation factors that
measure 250,000 daltons and 120,000 daltons in SDS (Figure 1 b and d
respectively). The remaining 5? of the gelation activity that was collected
in the 0-40% pellet was recovered with a complex of 5 low molecular weight
components that measure 48,000, 38,000, 32,000, 24,000 and 20,000 daltons in
SDS (Figure le). The polypeptides appear to be proteolytic breakdown
products of higher molecular weight gelation factors since their presence in
the extract was abolished by inclusion of proteolytic inhibtors.
Mapping the binding site of thymosin β4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin
