Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping.
The catalytic alpha-subunit of rat hepatic (Na+, K+)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody ("9-A5") covalently linked to Sepharose 4B that specifically blocks phosphorylation of the sodium pump's alpha-subunit from [gamma-32P]ATP [Schenk, D. B., Hubert, J.J., & Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951]. The hepatic subunit is virtually identical with purified rat, dog, and human renal alpha-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Mr 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal beta-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic "beta"-subunits (Mr 50K and 55K) eluted from 9-A5-Sepharose. Additional studies of ouabain-sensitive 86Rb+ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points (ID50 = 0.1 mM ouabain) in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two known structurally conserved forms of catalytic subunit (the renallike alpha form) and, if at all, structurally divergent forms of the sodium pump's beta-subunit. In addition, immunoaffinity chromatography with 9-A5-Sepharose facilitates the isolation of (Na+, K+)-ATPases from nonrenal tissues with low levels of sodium pumps.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007679:Kidney Medulla / Q000201:enzymology*; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D011916:Rats, Inbred F344; D011919:Rats, Inbred Strains; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium