Determination of Na(+)-K(+)-ATPase alpha- and beta-isoforms and kinetic properties in mammalian liver

• Published in: The American journal of physiology Vol. 262; no. 6 Pt 1; p. C1491
• Main Authors: Sun, Y, Ball, Jr, W J
• Format: Journal Article
• Language: English
• Published: United States 01.06.1992
• Subjects: Animals; Blotting, Western; Brain - enzymology; Cell Fractionation; Centrifugation, Zonal; Female; Gastric Mucosa - enzymology; Intestinal Mucosa - enzymology; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kidney Medulla - enzymology; Kinetics; Male; Microsomes - enzymology; Microsomes, Liver - enzymology; Organ Specificity; Rabbits; Rats; Rats, Inbred Strains; Sheep; Sodium-Potassium-Exchanging ATPase - isolation & purification; Sodium-Potassium-Exchanging ATPase - metabolism; Species Specificity
• ISSN: 0002-9513
• DOI: 10.1152/ajpcell.1992.262.6.C1491

While Western blot analysis clearly revealed the presence of the alpha- and beta-subunits of Na(+)-K(+)-ATPase in a variety of rat tissues, beta was not readily detectable in liver. This observation was consistent with a previous report indicating that Na(+)-K(+)-ATPase immunoprecipitated from rat liver gives no clear evidence for the presence of a beta-subunit (Hubert et al. Biochemistry 25: 4156-4163, 1986). However, Western blot analysis of density gradient-purified lamb and rat liver microsomes showed the presence of a protein with an approximate molecular mass of 42 kDa that was immunoreactive with beta-specific polyclonal antibodies as well as beta-directed monoclonal antibodies. Deglycosylation of this protein by N-glycosidase F generated a core protein (beta c, M(r) approximately 32,000) that had the identical electrophoretic mobility as the beta c protein of the purified kidney enzyme. Isoform-specific monoclonal and synthetic peptide-directed polyclonal antibodies were used to demonstrate the presence of only the alpha 1- and beta 1-proteins in the liver and the presence of beta 2 in rat brain. Functional studies then showed that although both rat and lamb liver enzymes had sensitivities to cardiac glycoside inhibition similar to that of their corresponding kidney enzyme, the lamb liver enzyme had higher affinities for Na+, K+, and ATP than the kidney enzyme.