Tubulin characterization during embryogenesis of Ascaris suum

Abstract

Tubulin in cytosolic fractions of Ascaris suum embryos was characterized on the basis of its specific colchicine binding and known properties of the tubulin-colchicine complex. Cytosolic fractions of early (eight-cell) and late (gastrula) embryos maintained at 37°C exhibited significant colchicine binding reaching pseudosaturation at 6 hr. No binding was detected in samples incubated for 8 hr at 0°C. Colchicine binding activity of late embryo cytosolic fractions in the absence of guanosine 5′-triphosphate or vinblastine sulfate decayed with first-order kinetics and had a t 1 2 of 377 min and a k of 1.84 × 10 −3 min −1. In the presence of 1 m M guanosine 5′-triphosphate ( t 1 2 = 563 min, k = 1.23 × 10 −3 min −1 ) or 0.5 m M vinblastine sulfate ( t 1 2 = 877 min, k = 0.79 × 10 −3 min −1 ), the tubulin-colchicine interaction was stabilized. Colchicine binding to late embryo tubulin was competitively inhibited by podophyllotoxin with a K i of 1.1 × 10 −6 M. The association constants of early and late soluble embryo tubulin for colchicine were 4.35 × 10 4 M −1 and 1.86 × 10 5 M −1, respectively. Although the affinity of early tubulin for colchicine was less than late embryo tubulin, the titratable soluble tubulin pools were equal in these stages. The tubulin pool was estimated to be 0.3% of the soluble embryo protein. The change in affinity of tubulin for colchicine during embryogenesis appeared to be unique to this organism. The importance of this change and the mechanisms involved in the regulation of tubulin affinities are discussed.