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)
Highlights
Na+,K+-ATPaseis digested by trypsin in the absence begins at ThreS4,i.e apparently the 19-kDa fragment of Ca2+and presence of Rb+ ions to a stable 19-kDa has been partially cut, without loss of Rb+ occlusion
We show that extensive diges- The experiments with trypsinP,ronase, and proteintion with nonselective fungal proteases (Pronase and ase K strongly suggest that Rb+occlusion sites are proteinase K) alone, in combination, or after tryptic located within transmembrane segments, including digestion can remove up to 70% of membrane protein those of the 19-kDa fragment and at least one of the without destroying Rb+ occlusion.In the most heavily smaller fragments
DigestionwithNonselective Proteases Compared to That with Trypsin-Digestion with the nonspecific proteases Pronase and Proteinase K removes up to 70% of the membrane protein without destroying Rb’ occlusion
Summary
Digestion with Trypsin, Pronase, or Proteinase K-Previ- minor bands, which is not surprising in viewof the lack of ously, we described the conditions for extensive tryptic diges- specificity of the Pronase. About 60% of the initial membrane tion at pH8.5 with retention of Rb+ occlusion As much as 70% of the protein was solubilized, and the specific activity essentially intact. Peptides of 10 kDa or less were not well resolved in remaining in the membrane after treatmentof Na+,K+-ATP-. Pronase alone (lane ing inthe membrane after treatment of Na+,K+-ATPase with P ) (1:lO enzyme:Pronase, w/w) removed >50% of the mem-. TPCK-trypsin (lune T ) , TPCK-trypsinandthenPronase brane protein including the bulk of both the CY and /3 chains (lane TP), Pronase (lane P ) , proteinase K (lane P K ) , and without destroying Rb’ occlusion (Table I). In some experiments using a higher ratio of enzyme to Pronase or
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