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 utilizing proteolytic digestion. Evidence is presented for a model with 10 transmembrane segments and lability of the C-terminal domain (M7-M10). Using reconstituted proteoliposomes, inside-out oriented pumps were digested with trypsin at the cytoplasmic surface. Evidence was obtained for the M7/M8 pair and cytoplasmic splits between M8 and M9 and between M9 and M10. Because an extracellular split between M9 and M10 was also observed, using right-side-out oriented renal microsomes, we propose that the M9/M10 pair either is destabilized by cytoplasmic digestion or is intrinsically mobile. Using renal microsomes, extracellular digestion 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 (beta-ME) and n-butanol. Both perturbations inactivate enzyme activity. Rb ions protect against inactivation and digestion. At 45 degrees C, with beta-ME and n-butanol, trypsin and chymotrypsin 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 surface, and an additional split between M8 and M9 is observed. Extracellular digestion of the alpha-subunit is associated with digestion of the beta-subunit near the first extracellular S-S bridge. Rb ions protect the beta-subunit. Exposure to proteases of extracellular domains of both subunits appears to be caused by disruption of subunit interactions.
Goldshleger
R
R
Biochemistry Department, Wiezmann Institute of Science, Rehovot, Israel.
Tal
D M
DM
Karlish
S J
SJ
D020001:1-Butanol; D000595:Amino Acid Sequence; D000818:Animals; D000440:Butanols / Q000737:chemistry; D003593:Cytoplasm / Q000201:enzymology; D006358:Hot Temperature; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D008623:Mercaptoethanol / Q000737:chemistry; D008861:Microsomes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011510:Proteolipids; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D000440:Butanols; D010446:Peptide Fragments; D011510:Proteolipids; C020485:proteoliposomes; D008623:Mercaptoethanol; D020001:1-Butanol; D000254:Sodium-Potassium-Exchanging ATPase