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

• Published in: The Journal of biological chemistry 1989-05, Vol.264 (14), p.8271-8280
• Main Authors: Urayama, O, Shutt, H, Sweadner, K J
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Animals, Newborn; Antibodies, Monoclonal - immunology; Antibody Specificity; Biological and medical sciences; brain; Brain - embryology; Brain - enzymology; Brain - growth & development; Cell-Free System; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Hydrolases; Isoenzymes - analysis; Isoenzymes - immunology; Isoenzymes - metabolism; Peptide Fragments - metabolism; Protein Biosynthesis; Rats; Rats, Inbred Strains; Serine Endopeptidases - metabolism; Sodium-Potassium-Exchanging ATPase - analysis; Sodium-Potassium-Exchanging ATPase - immunology; Sodium-Potassium-Exchanging ATPase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)83179-9

Molecular genetic evidence indicates that there should be three different (Na+ + K+)-stimulated ATPase (Na,K-ATPase) α subunit isozymes in the brain where previously only two (“α” and “α(+)”) were resolved as proteins. To detect and identify α1, α2, and α3 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 α1 comigrated with tissue α1, while α2 and α3 comigrated with the leading and trailing edges, respectively, of tissue “α(+).” Proteolytic fingerprints of newborn rat brain Na,K-ATPase labeled in vivo with L-[35S]methionine indicated the presence of α1 and α3, and a low level of α2. 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 α1 and α3 at different developmental ages, while α2 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.