Tryptic and chymotryptic cleavage sites in sequence of α-subunit of (Na + + K +)-ATPase from outer medulla of mammalian kidney

Abstract

Localization of selective proteolytic splits in α-subunit of (Na + + K +)-ATPase is important for understanding the mechanism of active Na +,K +-transport. Proteolytic fragments of α-subunit from pig kidney were purified by chromatography in NaDodSO 4 on TSK 3000 SW columns. NH 2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of α-subunit from sheep kidney (Shull, C.E., et al. (1985) Nature 316, 691–695) and pig kidney (Ovchinnikov, Y.A., et al. (1985) Proc. Acad. Sci. USSR 285, 1490–1495). The primary chymotryptic split in the E 1-form is located between Leu-266 and Ala-267 while the tryptic cleavage site appears to be between Arg-262 and Ile-263 (Bond 3). Tryptic cleavage in the initial fast phase of inactivation of the E 1-form is located between Lys-30 and Glu-31 (Bond 2). In the E 2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E 1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH 2-terminal for E 1-E 2 transition. Protonation of His-13 facilitates transition from E 1- to E 2-forms of the protein. Removal of His-13 after cleavage of bond 2 can explain the increase in apparent affinity of the cleaved enzyme for Na + and the shift in poise of E 1-E 2 equilibrium in direction of E 1-forms. The NH 2-terminal sequence in renal α-subunit is not conserved in α + from rat neurolemma or in α-subunit from Torpedo or brine shirmp. A regulatory function of the NH 2-terminal part of the α-subunit may thus be a unique feature of the α-subunit in (Na + + K +)-ATPase from mammalian kidney.