Occurrence of Spectrin-Like Protein in Y-l Adrenal Tumor Cells*

Abstract

With the aid of two monospecific antibodies raised in rabbits (antimouse erythrocyte spectrin and antimouse brain spectrin), the presence of a spectrin-like protein was demonstrated in mouse adrenal tumor (Y-l) cells. Y-l cells contain two large polypeptides, with mol wt characteristic of nonerythroid spectrin a- and β-subunits (240,000 and 235,000). When proteins from plasma membranes of Y-l cells were electrophoretically transferred to a nitrocellulose membrane, two polypeptides with mol wt of 240,000 and 225,000 were specifically stained with antimouse erythrocyte (rbc) spectrin immunoglobulin G (IgG). The rbc spectrin antibody was used to immuhoprecipitate Y-l spectrin from a neutral detergent (physiological ionic strength) cell extract. The 240,000 (α)- and 235,000 (β)- dalton polypeptides were immunoprecipitated in a 1:1 molar ratio, despite the fact that the antibody recognizes only the α-subunit. Two-dimensional chymotryptic peptide-mapping analysis indicated that the 240,000- and 235,000-dalton subunits of Y-l adrenal tumor spectrin are structurally unique and share limited homology with mouse rbc spectrin α- and β-subunits, but are nearly identical to the mouse brain spectrin 240,000- dalton a-subunit and 235,000-dalton β-subunit. Indirect immunofluorescence with anti-rbc or antibrain spectrin IgG and goat antirabbit IgG conjugated with rhodamine demonstrated intense staining at the plasma membrane and throughout the cytoplasm of Y-l cells, with little staining within the nucleus. (Endocrinology118: 2458–2463, 1986)