CRYPTIC NA+, K+-ATPASE ACTIVITY IN RAT-LIVER CANALICULAR PLASMA-MEMBRANES - EVIDENCE FOR ITS BASOLATERAL ORIGIN

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, glucagonstimulated adenyl cyclase, comigrated with this cryptic Na+, K+‐ATPase activity. Furthermore, addition of 6 μmol/L [12‐(2‐methoxyethoxy)‐ethyl‐8‐(cis‐2‐noctylcyclopropyl)‐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. Copyright © 1990 American Association for the Study of Liver Diseases