Actin in xenopus oocytes: II. intracellular distribution and polymerizability

Abstract

The largest oocytes of Xenopus laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions. The actin was identified in electrophoretograms by its MW and its binding to DNase I. Oocytes contain 1.4-1.7 .mu.g of actin per cell, of which up to 88% is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8% of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95% of the actin cannot be sedimented under forces that would pellet filamentous actin. Addition of K and Mg to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly 1/3 of the soluble actin is recovered from Sephadex columns at about the position of monomer. About 2/3 is in complexes of 100,000 daltons or greater.