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  • Title: Tryptic and chymotryptic cleavage sites in sequence of alpha-subunit of (Na+ + K+)-ATPase from outer medulla of mammalian kidney.
    Author: Jørgensen PL, Collins JH.
    Journal: Biochim Biophys Acta; 1986 Sep 11; 860(3):570-6. PubMed ID: 3017424.
    Abstract:
    Localization of selective proteolytic splits in alpha-subunit of (Na+ + K+)-ATPase is important for understanding the mechanism of active Na+,K+-transport. Proteolytic fragments of alpha-subunit from pig kidney were purified by chromatography in NaDodSO4 on TSK 3000 SW columns. NH2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of alpha-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 E1-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 E1-form is located between Lys-30 and Glu-31 (Bond 2). In the E2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH2-terminal for E1-E2 transition. Protonation of His-13 facilitates transition from E1- to E2-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 E1-E2 equilibrium in direction of E1-forms. The NH2-terminal sequence in renal alpha-subunit is not conserved in alpha + from rat neurolemma or in alpha-subunit from Torpedo or brine shrimp. A regulatory function of the NH2-terminal part of the alpha-subunit may thus be a unique feature of the alpha-subunit in (Na+ + K+)-ATPase from mammalian kidney.
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