Papain fragmentation of the sodium-potassium ATPase .beta. subunit reveals multiple membrane-bound domains

Abstract

Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2-terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain.

Primary structure of the β‐subunit of Torpedo californica (Na+ + K+)‐ATPase deduced from the cDNA sequence

“…The 305th codon specifying serine is followed by 2 successive translational termination codons (TGA and TAA). Thus, it is concluded that the&subunit of T. cafifornica (Na+ + K+)-ATPase consists of 305 amino acid residues (including the initiating methionine) and has a calculated M, of amino-terminal sequence of the lamb [21] or dog 34671, which agrees with the reported Mr of the kidney (Na+ + K+)-ATPase P-subunit [22]; in the protein moiety of this subunit (32000-38000) latter case, residue 12 or 13 in the Torpedo se-[4,17-201. The sequence of amino acid residues quence is assumed to be an insertion.…”

“…On the basis of these observations, it is assumed that the large portion of the P-subunit molecule following the putative transmembrane segment is located on the extracellular side of the membrane, whereas the small amino-terminal portion resides on the cytoplasmic side. This transmembrane topology of the P-subunit is consistent with previous findings as follows: (i) immunochemical [33] and chemical labelling studies [34] indicate that the p-subunit spans the membrane; (ii) chemical labelling [35,36] and cell-free translation studies [19] indicate that virtually all of the hydrophilic part of the Psubunit is located on the extracellular side of the membrane; (iii) studies by pulse-chase labelling and endoglycosidase digestion suggest that the ,& subunit has 2 or more N-linked oligosaccharide chains [20,37]; (iv) the results of papain fragmentation suggest that the carboxy-terminal fragment of M, 16000 as well as the preceding fragment of M, 3 1000 contains covalently bound carbohydrate, whereas the amino-terminal fragment of M, 9000 apparently has no carbohydrate [22]. Three additional segments comprising almost exclusively uncharged residues (residues 201-217, 231-249 and 255-272) are present in the carboxyterminal region of the &subunit.…”

Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions

“…Additions to the reaction mixture are detailed in the figure legend. Incorporation was quantitated by solubilizing the gel slices (Chin, 1985) for scintillation counting.…”

Pig kidney Na+,K+‐ATPase

“…The connection between transmembrane rods is, apparently, exposed on the membrane cytoplasmic side. Only this region can enclose the papain split site of the P-subunit that results in two large membrane-bound glycosylated fragments [42]. The hydrophilic character of the P-subunit Cterminal part demonstrates its extracellular orientation.…”