Isolation and biochemical characterization of ten-nanometer filaments from cultured ovarian granulosa cells.

Abstract

Ten-nm filaments have been isolated from control and colchicine-treated primary cultures of rat ovarian granulosa cells. Negative stain electron microscopy indicates an average filament diameter of 10.3 nm in the isolated fiber bundles, which, upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, are found to contain a major polypeptide with a molecular weight of 57,000 and several minor components including actin. One-dimensional peptide mapping and two-dimensional gel electrophoresis demonstrate similarity between the granulosa cell and baby hamster kidney cell 10-nm filament subunit protein, both of which are distinguishable from keratin, desmin, actin, and tubulin. Quantitative gel densitometry experiments demonstrate little difference in the total amount of the 10-nm filament protein in control cells or cells treated with colchicine, accounting for 12 or 15% of the total cellular protein, respectively. The purification procedure, which involves extraction in Triton X-100 and KCl followed by DNase I treatment, yields 709% of the total granulosa cell intermediate filament protein, and 70% of the newly synthesized 57,000 molecular weight component. Two-dimensional gel electrophoresis of cultures labeled with [32P]phosphate show by autoradiography that the 57,000-dalton polypeptide, actin, and a 130,000-dalton protein are the most readily phosphorylated polypeptides in granulosa cell cultures. These studies identify the major intermediate filament subunit protein of granulosa cells as a 57,000-dalton phosphorylatable polypeptide which comprises a substantial portion of the granulosa cell cytoskeleton.