TOPOLOGY OF THE ALPHA-SUBUNIT OF NA,K-ATPASE BASED ON PROTEOLYSIS - LABILITY OF THE TOPOLOGICAL ORGANIZATION

Topology of the alpha-subunit of Na,K-ATPase has been analyzed utilizi ng proteolytic digestion. Evidence is presented for a model with 10 tr ansmembrane segments and lability of the C-terminal domain (M7-M10), U sing reconstituted proteoliposomes, inside-out oriented pumps were dig ested with trypsin at the cytoplasmic surface, Evidence was obtained f or the M7/M8 pair and cytoplasmic splits: between M8 and M9 and betwee n M9 and M10. Because an extracellular split between M9 and M10 was al so observed, using right-side-out oriented renal microsomes, we propos e that the M9/M10 pair either is destabilized by cytoplasmic digestion or is intrinsically mobile, Using renal microsomes, extracellular dig estion of the alpha-subunit by trypsin, chymotrypsin, or an endogenous protease has been observed, after incubation at 55 or at 45 degrees C with beta-mercaptoethanol CB-ME) and n-butanol, Both perturbations in activate enzyme activity. Rb ions protect against inactivation and dig estion. At 45 degrees C, with beta-ME and n-butanol, trypsin and chymo trypsin cut between M7 and M8 and between M9 and M10, consistent with the 10-segment model, At 55 degrees C, the topological organization is altered, the M8/M9 connecting loop is exposed at the extracellular su rface, and an additional split between Ms and M9 is observed. Extracel lular digestion of the alpha-subunit is associated with digestion of t he beta-subunit near the first extracellular S-S bridge. Rb ions prote ct the beta-subunit. Exposure to proteases of extracellular domains of both subunits appears to be caused by disruption of subunit interacti ons.