Identification of a membrane-embedded segment of the large polypeptide chain of (Na⁺, K⁺)ATPase

Abstract

THE sodium pump or (Na⁺, K⁺)ATPase couples hydrolysis of ATP to the active transport of Na⁺ and K⁺ across the cell membrane. The firm association of the enzyme with the membrane allows its purification from the outer renal medulla by selective extraction of other membrane proteins using sodium dodecyl sulphate (SDS)¹. Two polypeptide components remain in the purified preparation of (Na⁺, K⁺)ATPase. A catalytic protein with molecular weight (MW) about 100,000 (100 K) which constitutes 60–70% of the total protein and a smaller sialoglycoprotein of unknown function^2,3. The arrangement of these proteins within the membrane is an important but poorly understood aspect of the Na pump structure. Presumably the catalytic regions of the enzyme are in contact with the aqueous phase at the membrane surface, while other portions of the protein are in intimate contact with the lipid and account for its firm anchorage. The lipid associated domains of the pump are of special interest because in addition to a purely structural role, they may also form parts of the ion conducting pathways. We have been able to identify presumptive intra-membranous portions of the Na pump protein by labelling them covalently from within the membrane with 5-[¹²⁵I]-iodo-naphthyl-1-azide (INA). This label is a very hydrophobic light-sensitive compound developed previously for this purpose and tested with other systems^4–7. A segment(s) of approximate MW 12K derived from the large polypeptide chain has been identified by combining the INA-labelling technique with extensive proteolysis of the membrane bound enzyme. Graded proteolysis of the labelled (Na⁺, K⁺)ATPase has also permitted an estimate of the position of this segment within the large chain.