Purification and immunochemical properties of human Na,K-ATPase alpha subunits and formic acid-derived polypeptide...

In this study, alpha (alpha) isoform proteins were purified from the partially purified Na,K-ATPase by SDS-PAGE and electroelution. Peptide mapping showed subtle biochemical differences between alpha subunit proteins of rat and human origin. The purified alpha 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 alpha proteins (R alpha) 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. R alpha and R40 were successfully used for immunohistochemical staining of fibers in the white matter of the human brain frontal cortex. These antisera were not isoform-specific, they cross-reacted with 40, 50, and 60 kDa polypeptides as well as the three alpha bands.