Photoaffinity labeling of the ouabain binding site in Na, K-ATPase in developing brine shrimp.
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.
D000345:Affinity Labels; D000818:Animals; D001156:Artemia / Q000201:enzymology*; D001665:Binding Sites; D003979:Diazonium Compounds / Q000378:metabolism; D004073:Digitoxigenin / Q000031:analogs & derivatives* / Q000378:metabolism; D004074:Digitoxin / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010042:Ouabain / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D000345:Affinity Labels; D003979:Diazonium Compounds; D004073:Digitoxigenin; D010042:Ouabain; C027274:4'''-(2-ethyldiazomalonyl)digitoxin; C043225:4'-(2-ethyldiazomalonyl)digitoxigenin monodigitoxiside; D004074:Digitoxin; D000254:Sodium-Potassium-Exchanging ATPase