Occurrence of spectrin-like protein in Y-1 adrenal tumor cells

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-1) cells. Y-1 cells contain two large polypeptides, with mol wt characteristic of nonerythroid spectrin alpha- and beta-subunits (240,000 and 235,000). When proteins from plasma membranes of Y-1 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 immunoprecipitate Y-1 spectrin from a neutral detergent (physiological ionic strength) cell extract. The 240,000 (alpha)- and 235,000 (beta)-dalton polypeptides were immunoprecipitated in a 1:1 molar ratio, despite the fact that the antibody recognizes only the alpha-subunit. Two-dimensional chymotryptic peptide-mapping analysis indicated that the 240,000- and 235,000-dalton subunits of Y-1 adrenal tumor spectrin are structurally unique and share limited homology with mouse rbc spectrin alpha- and beta-subunits, but are nearly identical to the mouse brain spectrin 240,000-dalton alpha-subunit and 235,000-dalton beta-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-1 cells, with little staining within the nucleus.