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

Abstract

O-Hexanoyl-3,5-diiodo-N-(4-azido-2-nitro-phenyl)tyramine has been used after photochemical conversion into the reactive nitrene to label (Na+,K+)-ATPase from Bufo marinus toad kidney. Immunochemical evidence indicates that the reagent labels both subunits of the enzyme in partially purified form as well as in microsomal membranes. These results support the view that the glycoprotein subunit, like the catalytic subunit, possesses hydrophobic domains by which it is integrated into the plasma membrane.

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.…”

Pig kidney Na+,K+‐ATPase

“…Modification of Na+,K+-ATPase with permeable hydrophobic reagents [39,40] demonstrates that a part of the Psubunit is buried in the lipid bilayer. Labelling of the exposed enzyme portion with fluorescamine [7-111 or Bolton-Hunter reagent [36] and immunochemical assay [5] showed that practically the whole hydrophilic part of the P-subunit is exposed on the outer surface of the membrane.…”

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit.

“…A binding site for cardiac glycosides, such as ouabain, is located on the extracytoplasmic side while both the ATP-binding site and the phosphorylation site are located on the cytoplasmic side of the ct subunit. Contrary to the ot subunit which appears to lack covalently bound carbohydrates, the/3 subunit is a glycosylated transmembrane protein whose principal mass protrudes into the intercellular space (Girardet et al, 1983;Noguchi et al, 1986;Shull et al, 1986;Ovchinnikov et al, 1986;Brown et al, 1987). The two enzyme subunits are synthesized independently from separate mRNA species (Geering et al, 1985).…”