Purification and Immunochemical Properties of Human Na, K-ATPase Alpha Subunits and Formic Acid-Derived Polypeptide Fragments

Abstract In this study, alpha (α) isoform proteins were purified from the partially purified Na, K-ATPase by SDS-PAGE and electrocution. Peptide mapping showed subtle biochemical differences between α subunit proteins of rat and human origin. The purified α proteins were treated with formic acid, the cleaved polypeptide fragments were separated by SDS-PAGE, the bands corresponding to 40, 50, and 60 kDa were excised, and the proteins were electroeluted. The purified 40, 50, and 60 kDa polypeptides were essentially homogeneous, and were used for preparation of polyclonal antibodies in rabbits. The antisera to α proteins (Rα) and 60 & 40 kDa polypeptides (R60 & R40) were obtained and characterized by Western blotting. All three antisera were highly specific, since they cross-reacted with only the 100 kDa bands of the crude brainstem homogenates, of the axolemma, and of the cerebral cortex synaptosomes and microsomes. Rα and R40 were successfully used for immunohistochemical staining of fibers in the white ma...