HEPATIC NA-K+-ATPASE ENZYME-ACTIVITY CORRELATES WITH POLARIZED BETA-SUBUNIT EXPRESSION()

We have examined underlying causes for observations made in hepatocyte s in which catalytic subunits of Na+-K+-ATPase are found both in bile canalicular (apical) and sinusoidal (basolateral) membrane domains, wh ereas functional activity is associated preferentially with sinusoidal membrane sites. In a series of parallel studies, we determined by bot h light and electron microscopy that Na+-K+-ATPase alpha-subunits were localized to both membrane domains of hepatocytes. With the use of pu rified liver plasma membrane subfractions, ouabain inhibition curves d emonstrated similar inhibition constants (inhibition constant 10(-5) M ), and immunoblots using alpha(1)-, alpha(2)-, alpha(3)-polyclonal and monoclonal antibodies demonstrated antigenic sites predominantly for alpha(1) in both membrane fractions. Also, Northern blot hybridization analysis revealed only the alpha(1)-isoform in hepatocytes. In contra st to the bipolar distribution of the alpha(1)-subunit, the beta-subun it was identified only at the sinusoidal surface using fluorescence la beling with a monoclonal antibody. The beta(1)-isoform was demonstrate d by Northern blot analysis and was present predominantly at the sinus oidal domain by immunoblotting with polyclonal antibodies. In addition to the bipolar distribution of oil, immunoblotting of liver plasma me mbrane subfractions demonstrated a symmetrical distribution of fodrin, ankyrin, actin, and E-cadherin at both domains. These results suggest that functionally competent alpha/beta-complexes form at the sinusoid al domain, whereas only alpha(1)-subunits are present at the apical po le.