Labelling of the hydrophobic domain of the Na⁺,K⁺‐ATPase

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

“…Studies using hydrophobic photoactivatcd reagents support the existence of membrane-embedded domains of the 0 subunit (Giradet et al, 1983;Jorgensen & Brunner, 1983;Montecucco et al, 1981). Giradet et al (1981) have generated a 0-subunit antiserum which still binds to toad kidney microsomes after absorption with intact toad erythrocytes.…”

“…This model is presented as a framework for future experiments. It would be interesting to determine the partitioning of the lipid-soluble and digitoxin labels (Giradet et al, 1983;Jorgensen & Brunner, 1983;Montecucco et al, 1981;Farley et al, 1980;Hall & Ruoho, 1980) among the papain fragments.…”

“…brough & Bayne, 1983; Craig & Kyte, 1980). Lipid-soluble reagents and a digitoxin derivative have been shown to react with the 8 subunit (Jorgensen & Brunner, 1983;Jorgensen et al, 1982; Montecucco et al, 1981;Farley et al, 1980;Hall & Ruoho, 1980). Giradet et al (1981) have described an antiserum apparently directed toward cytoplasmic determinants of the 8 subunit.…”

1-Palmitoyl-2-(p-benzoyl)benzoyl phosphatidylcholine, a photoactive phospholipid for the labelling of membrane components

“…Snake-venom phospholipase A2 and cabbage phospholipase D were the products of Boehringer (Mannheim, Germany). Sarcoplasmic-reticulum Ca2+activated ATPase and Electrophorus electricus (Na++ K+)-activated ATPase were prepared and assayed as described previously Montecucco et al, 1981). Protein concentration was determined by the Lowry method, with bovine serum albumin as standard, and phospholipid phosphorus as described by Bartlett (1959).…”

Hydrophobic labeling of (sodium, potassium)-ATPase: further evidence that the .beta. subunit is embedded in the membrane bilayer

“…One approach to such questions consists of the use of lipophilic affinity labeling reagents which are generated photochemically within the membrane and hence allow the labeling and characterization of regions of membrane polypeptide embedded in the lipid bilayer (Chowdhry & Westheimer, 1979), Thus, a hydrophobic domain of the a subunit of purified (Na+,K+)-ATPase has been clearly identified by the use of lipophilic nitrenes or carbenes (Karlish et al, 1977;Farley et al, 1980;Jorgensen et al, 1982). In addition, the 0 subunit appears to possess a hydrophobic domain since when (Na+,K+)-ATPase preparations were treated with photoreactive phosphatidylcholine analogues both 95-and 45-dalton polypeptides of the purified enzyme were labeled (Montecucco et al, 1981). However, this does not provide rigorous proof for the tight association of the 0 subunit with cellular membranes since the labeling was performed in the presence of detergents required for the purification of the enzyme.…”