Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain.

Molecular genetic evidence indicates that there should be three different (Na+ + K+)-stimulated ATPase (Na,K-ATPase) alpha subunit isozymes in the brain where previously only two ("alpha" and "alpha(+)") were resolved as proteins. To detect and identify alpha 1, alpha 2, and alpha 3 isozymes, polypeptides made by cell-free translation (Schneider, J.W., Mercer, R.W., Gilmore-Hebert, M., Utset, M.F., Lai, C., Greene, A., and Benz, E.J., Jr. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 284-288) were analyzed by gel electrophoresis and proteolytic fingerprinting. Synthetic alpha 1 comigrated with tissue alpha 1, while alpha 2 and alpha 3 comigrated with the leading and trailing edges, respectively, of tissue "alpha(+)." Proteolytic fingerprints of newborn rat brain Na,K-ATPase labeled in vivo with L-[35S]methionine indicated the presence of alpha 1 and alpha 3, and a low level of alpha 2. Monoclonal antibodies were characterized by the electrophoretic mobility of their antigens and by their ability to recognize the Na,K-ATPases of kidney, brain, and skeletal muscle. The antibodies were used to assess isozyme expression in the brain. All three isozymes increased in abundance during development from the 18-day fetus to the adult. Small changes were seen in the relative level of expression of alpha 1 and alpha 3 at different developmental ages, while alpha 2 expression increased markedly between the neonate and adult. In adult brain, all three isozymes were found in all brain regions examined. We conclude that all three isozymes are expressed as proteins and that their expression and distribution must be under complex control. No single developmental age or macroscopic brain region provides an exclusive source of any of the isozymes.