title: Cryptic Na+,K(+)-ATPase activity in rat liver canalicular plasma membranes: evidence for its basolateral origin.
abstract: Controversy exists concerning the localization of the enzyme Na+,K(+)-ATPase to canalicular membranes in hepatocytes. Most studies find enzyme activity only at the basolateral plasma membrane domain of the hepatocyte. However, Na+,K(+)-ATPase activity has been detected recently in a canalicular membrane fraction prepared by Mg++ precipitation, suggesting that differences in membrane domain fluidity account for these discrepancies. To reinvestigate this question, we used free-flow electrophoresis to further purify canalicular liver plasma membranes originally separated by sucrose density centrifugation. With this technique, canalicular membranes devoid of Na+,K(+)-ATPase activity by routine assay were separated into six subfractions. More than 80% of the activities of canalicular marker enzymes was recovered in two subfractions closest to the anode, which were totally devoid of Na+,K(+)-ATPase activity. However, Na+,K(+)-ATPase activity could now be detected in the four other fractions that contained only small amounts of canalicular marker enzymes. The basolateral marker enzyme, glucagon-stimulated adenyl cyclase, comigrated with this cryptic Na+,K(+)-ATPase activity. Furthermore, addition of 6 mumol/L [12-(2-methoxyethoxy)-ethyl-8-(cis-2-n-octylcyclopropyl)-octanoate ], a membrane-fluidizing agent, to the original canalicular membrane preparation and to all subfractions did not stimulate or unmask latent Na+,K(+)-ATPase activity. Finally, when canalicular membranes isolated by Mg++ precipitation were subjected to free-flow electrophoresis, they could not be separated from the more positively charged Na+,K(+)-ATPase-containing fractions, probably because of alterations in surface charge. Together these findings suggest that Na+,K(+)-ATPase is a basolateral enzyme, that represents a small contaminant when present in canalicular liver plasma membranes and that methodological differences may account for previous discrepancies.
authors.affiliation: Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510.
authors.forename: M
authors.initials: M
authors.lastname: Sellinger
authors.forename: C
authors.initials: C
authors.lastname: Barrett
authors.forename: P
authors.initials: P
authors.lastname: Malle
authors.forename: E R
authors.initials: ER
authors.lastname: Gordon
authors.forename: J L
authors.initials: JL
authors.lastname: Boyer
mesh_terms: D000818:Animals; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000494:pharmacology; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000648:ultrastructure; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008560:Membrane Fluidity / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013228:Stearates / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature
chemical_list: D001592:Benzyl Alcohols; D013228:Stearates; C023714:A(2)C; D017301:Ca(2+) Mg(2+)-ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D019905:Benzyl Alcohol