Chymotryptic cleavage of α-subunit in E 1-forms of renal (Na + + K +)-ATPase: effects on enzymatic properties, ligand binding and cation exchange

Abstract

Chymotrypsin in NaCl medium at low ionic strength rapidly cleaves a bond in the N-terminal half of the α-subunit of pure membrane-bound (Na + + K +)-ATPase from outer renal medulla. Secondary cleavage is very slow and the α-subunit can be converted almost quantitatively to a 78 kDa fragment. The sensitive bond is exposed to cleavage when the protein is stabilized in the E 1 form by binding of Na + or nucleotides. The bond is protected in medium containing KCl (E 2 K form), but it is exposed when ADP or ATP are added (E 1KATP form). Fluorescence analysis and examination of ligand binding and enzymatic properties of the cleaved protein demonstrate that cleavage of the bond stabilizes the protein in the E 1 form with sites for tight binding of nucleotides and cations exposed to the medium. About two 86Rb ions are bound per cleaved α-subunit with normal affinity ( K d = 9 μM ). The bound Rb + is not displaced by ATP or ADP. The nucleotide-potassium antagonism is abolished and ATP is bound with high affinity both in NaCl and in KCl media. Na +-dependent phosphorylation is quantitatively recovered in the 78 kDa fragment, but the affinity for binding of [ 48V]vanadate is very low after cleavage. ADP-ATP exchange is stimulated 4–5-fold by cleavage; while nucleotide dependent Na +-Na +, K +-K +, or Na +-K + exchange are abolished. Cleavage with chymotrypsin in NaCl at the N-terminal side of the phosphorylated residue thus stabilizes the E 1 form of the protein and abolishes cation exchange and conformational transitions in the protein although binding of cations, nucleotides and phosphate is preserved. In contrast, cleavage with trypsin in KCl at the C-terminal side of the phosphorylated residue does not interfere with E 1-E 2 transitions and Na +-Na + or K +-K + exchange. This data support the notion that cation exchange and E 1–E 2 transitions are thightly coupled.