Separation and interaction of the major components of sea urchin actin gel.

Abstract

Abstract Warming of low calcium, isotonic extracts of sea urchin eggs causes gelation by inducing the polymerization of F-actin which then interacts with other proteins in the cytosol to form the gel. Two major protein components with molecular weights of 58,000 and 220,000 have been identified. F-actin has previously been purified from such preparations and shown to interact with a mixture of these proteins to form a gel composed of F-actin aggregates displaying a unique 11 nm banding pattern. The gelation is calcium-sensitive; half-maximal inhibition is at 1 to 2 μ m free calcium in the extract. Solubilization of the gel in 0.6 m -KI and chromatography on A-15m agarose in 0.6 m -KCI resolves the 220K protein (Mr = 220,000) from G-actin and the 58K protein (Mr = 58,000). Further purification on DEAE-cellulose yields pure 58K protein. When combined, the repolymerized actin and 58K protein form microscopic needles composed of linear aggregates of F-actin with the characteristic 11 nm banding. Saturation of the 58K bindingsites of F-actin occurs at ratios of one 58K protein/4 to 5 actin molecules. This actin—58K protein interaction does not show calcium sensitivity. Addition of the 220K protein to pure F-actin produces no detectable aggregation and this protein therefore appears to be distinct from the high molecular weight, actin-binding proteins isolated from vertebrates. The addition of the 220K protein to solutions of needles causes aggregation to a material morphologically indistinguishable from the original gel, but this gelation is not sensitive to calcium. These results show that the 58K protein links F-actin filaments to form cross-banded needles with a 11 nm periodicity, which are then linked into a three-dimensional gel by the 220K protein. Theso results suggest that the initial calcium sensitivity may be due to the presence of regulatory proteins which are removed during the purification.