Photoaffinity labeling of the ouabain binding site in Na, K‐ATPase in developing brine shrimp

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Abstract
Analysis of purified Na,K‐ATPase from brine shrimp nauplii by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis reveals two large (α) subunits [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The band with lower mobility in a neutral or alkaline gel is designated α1 and the band with higher mobility α2. Ouabain prevents dephosphorylation of both α1 and α2 as documented by gel analysis, but a higher concentration of ouabain is required to prevent dephosphorylation of α2. The photoaffinity label, [3H]4′(2‐ethyldiazomalonyl) digitoxigenin monodi‐gitoxiside, specifically labels α in a ouabain‐protectable manner without labeling other contaminating proteins in the preparation. Greater than 93% of the total ouabain‐protectable labeling of the α subunits is associated with α1. The photoaffinity label, [3H]4‴ (2‐ethyldiazomalonyl) digitoxin, specifically labels α1 and β in a ouabain‐protectable manner without labeling other contaminating proteins. These data show that in the brine shrimp the third digitoxose residue of digitoxin binds in a region in which the α1 and β chains are in close proximity. Less than 5% of the specific ouabain‐protectable labeling of total α is associated with α2. These studies indicate that cardioactive steriods have higher affinity for the α1 subunit.