Asymmetric orientation of amino groups in the α-subunit and the β-subunit of (Na + + K +)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

Abstract

The orientation of amino groups in the membrane in the α- and β-subunits of (Na + + K +)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[ 125I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na + + K +)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na + + K +)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of 125I-labeling within the α-subunit. The preservation of (Na + + K +)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E 1- and E 2-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.