Immunochemical evidence for a transmembrane orientation of both the (sodium(1+), potassium(1+) ion-activated)-ATPase subunits

Abstract

Antibodies were raised against the large catalytic subunit (apparent Mr 96000) and the glycoprotein (apparent Mr 60000) of the sodium- and potassium-dependent adenosine triphosphatase [(Na+, K+)-ATPase] from Bufo marinus. The specificity of each antiserum was assessed by two-dimensional immunoelectrophoresis using toad kidney microsomes or the purified holoenzyme as a source of antigen and by indirect immunoprecipitation of detergent-solubilized (Na+, K+)-ATPase subunits from radioiodinated or biosynthetically labeled kidney holoenzyme, microsomes, or postnuclear supernatant. The anticatalytic subunit serum reacted exclusively with a 96000-dalton protein. The antiserum to the glycoprotein was rendered specific to this subunit by absorption with purified catalytic subunit. The two antisera were agglutinating and lytic in the presence of complement when toad erythrocytes were used as targets, indicating that antigenic determinants of both subunits were exposed on the cell surface. The specific reactivities with surface-exposed antigenic determinants of both subunits could be absorbed with toad red blood cells. Such absorbed antisera still reacted with detergent-treated or untreated kidney microsomes, revealing the presence of cytoplasmic and/or intramembranous antigenic sites. Our immunochemical data demonstrate that the glycoprotein subunit of (Na+, K+)-ATPase spans the lipid bilayer and confirm the transmembrane orientation of the catalytic subunit postulated from functional studies.

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

“…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.…”

Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and C Modulates Distinct Na,K-ATPase Isozymes

“…Membranes were blocked with 10% nonfat dried milk in Trisbuffered saline containing 0.1% Tween 20 and incubated with primary antibody. The following primary antibodies were used: 1) a PLM antibody recognizing PLM, which is not phosphorylated at Ser 68 (10) (1:500) (kindly provided by H. Garty and S. Karlish, Weizmann Institute, Israel) (we show in this study that this antibody recognizes the S68A and S68D PLM mutants, suggesting that it is the phosphate moiety that impedes recognition of PLM by this antibody); 2) a PLM antibody CP68 recognizing PLM phsphorylated at Ser68 (28) (1/10Ј000) (kindly provided by D. M. Bers (Loyola University, Chicago, IL)); and 3) a Na,K-ATPase ␣ subunit antibody (1:10,000) (27). After binding of the primary antibody, peroxidase-coupled secondary antibodies (1:10,000; Amersham Biosciences) were bound, and the complex was revealed with the ECL chemiluminescence kit (Amersham Biosciences) according to the manufacturer's protocol.…”

“…To study protein expression and PLM association, cRNA-injected oocytes were incubated in modified Barth's solution in the presence of 1 mCi/ml [ 35 S]methionine (Easy Tag Express [ 35 S]Protein Labeling Kit; PerkinElmer Life Sciences) for 6 h and subjected to 24-and 48-h chase periods in modified Barth's solution containing 10 mM unlabeled methionine. After the pulse and chase periods, microsomes were prepared and subjected to immunoprecipitations under nondenaturing conditions as described (26) with an antibody against the Na,K-ATPase ␣ subunit (27), and proteins were resolved by SDS-PAGE and revealed by fluorography.…”

Structural and Functional Interaction Sites between Na,K-ATPase and FXYD Proteins

“…Immunoprecipitation and Quantification of Association between FXYD Proteins and Na,K-ATPase-FXYD2 and FXYD4 were co-immunoprecipitated with wild type or mutant Na,K-ATPase ␣1 subunits under non-denaturing conditions as described (24) by using an ␣1 subunit antibody (26). Since co-immunoprecipitation of FXYD7 by an ␣ subunit antibody is not very efficient, the association of FXYD7 with Na,K-ATPase was tested by using an FXYD7 antibody (17) under nondenaturing conditions.…”