title: Extensive digestion of Na+,K(+)-ATPase by specific and nonspecific proteases with preservation of cation occlusion sites.
abstract: This paper extends our recent report that renal Na+,K(+)-ATPase is digested by trypsin in the absence of Ca2+ and presence of Rb+ ions to a stable 19-kDa fragment and smaller membrane-embedded fragments of the alpha chain and essentially intact beta chain. These are referred to as "19-kDa membranes." Occlusion of both Rb+ (K+) or Na+ ions is preserved, but ATP-dependent functions are lost (Karlish, S. J. D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570). We now show that extensive digestion with nonselective fungal proteases (Pronase and proteinase K) alone, in combination, or after tryptic digestion can remove up to 70% of membrane protein without destroying Rb+ occlusion. In the most heavily digested membranes, the 19-kDa fragment or a slightly shorter 18.5-kDa fragment and smaller fragments of the alpha chain remain, whereas the beta chain is largely digested, leaving smaller membrane-embedded fragments (13-15 kDa). For either trypsin or Pronase digestion, preservation of Rb+ occlusion and the specific fragmentation pattern is observed only in the absence of divalent metal ions (Mg2+ or Ca2+) and presence of either Rb+ or Na+ or congener ions. Tryptic digestion at pH 7.0 can split the beta chain into two fragments of approximately 50 and 16 kDa joined by an S-S bridge. The 16-kDa fragment is protected against further digestion by the presence of Rb+ ions, but probably is not directly involved in occluding cations. Tryptic 19-kDa membranes show a clear and reproducible fragmentation pattern in which all predicted membrane segments are identifiable. Families of fragments from 19-kDa membranes, including seven peptides of 7.6-11.7 kDa, have been separated by size-exclusion high performance liquid chromatography, concentrated, and resolved on 16.5% Tricine gels. N-terminal sequences of the different fragments have been determined after transfer to polyvinylidene difluoride paper. The most interesting findings are as follows. (a) Whereas the 19-kDa tryptic fragment begins at Asn831 as reported previously, the 18.5-kDa Pronase fragment begins at Thr834. (b) Fragments in tryptic 19-kDa membranes of 7.6-11.7 kDa begin at Asp68, Ile263, and Gln737, respectively. These include all putative transmembrane segments other than those in the 19-kDa fragment. (c) A Pronase fragment of 7.8 kDa begins at Thr834, i.e. apparently the 19-kDa fragment has been partially cut, without loss of Rb+ occlusion. (d) Tryptic 16- and approximately 50-kDa fragments of the beta chain begin at Ala5 and Gly143, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: J M
authors.initials: JM
authors.lastname: Capasso
authors.forename: S
authors.initials: S
authors.lastname: Hoving
authors.forename: D M
authors.initials: DM
authors.lastname: Tal
authors.forename: R
authors.initials: R
authors.lastname: Goldshleger
authors.forename: S J
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D006868:Hydrolysis; D007679:Kidney Medulla / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism; D011402:Pronase / Q000378:metabolism*; D012413:Rubidium / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D002412:Cations; D008565:Membrane Proteins; D012964:Sodium; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D011402:Pronase; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium; D002118:Calcium