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

✍ Scribed by Churchill, Lynn ;Hall, Clifford C. ;Peterson, Gary L. ;Ruoho, Arnold E. ;Hokin, Lowell E.

• Publisher: John Wiley and Sons
• Year: 1984
• Tongue: English
• Weight: 665 KB
• Volume: 231
• Category: Article
• ISSN: 0022-104X
• DOI: 10.1002/jez.1402310307

✦ Synopsis

Analysis of purified Na,K-ATPase from brine shrimp nauplii by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large (alpha) 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 alpha 1 and the band with higher mobility alpha 2. Ouabain prevents dephosphorylation of both alpha 1 and alpha 2 as documented by gel analysis, but a higher concentration of ouabain is required to prevent dephosphorylation of alpha 2. The photoaffinity label, [3H]4'(2-ethyldiazomalonyl) digitoxigenin monodigitoxiside, specifically labels alpha in a ouabain-protectable manner without labeling other contaminating proteins in the preparation. Greater than 93% of the total ouabain-protectable labeling of the alpha subunits is associated with alpha 1. The photoaffinity label, [3H]4"' (2-ethyldiazomalonyl) digitoxin, specifically labels alpha 1 and beta 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 alpha 1 and beta chains are in close proximity. Less than 5% of the specific ouabain-protectable labeling of total alpha is associated with alpha 2. These studies indicate that cardioactive steroids have higher affinity for the alpha 1 subunit.