Labeling of the glycoprotein subunit of (Na,K)ATPase with fluorescent probes.
Sodium plus potassium activated adenosinetriphosphatase [(Na,K)ATPase] is composed of a catalytic subunit (alpha) and a glycoprotein subunit (beta) of unknown function. A method has been developed to label the beta subunit of purified dog kidney (Na,K)ATPase with fluorescent probes. The method consists of oxidation of beta-subunit oligosaccharides, reaction of the resulting aldehydes with fluorescent hydrazides, and reduction of the hydrazones and unreacted aldehydes with NaBH4. Two oxidation methods were compared. Simultaneous treatment with neuraminidase and galactose oxidase did not inhibit significantly (Na,K)ATPase activity and allowed insertion of up to 11 mol of probe per mol of beta. In contrast, oxidation of (Na,K)ATPase oligosaccharides with periodate resulted in 50-80% inhibition of the (Na,K)ATPase activity with low or undetectable labeling. Eleven commercial probes and two novel hydrazides were tested for labeling of (Na,K)ATPase treated with galactose oxidase and neuraminidase. Eight probes did not label (Na,-K)ATPase but labeled red cell ghosts oxidized with periodate. Four probes labeled beta specifically but either adsorbed to the membrane tightly, or cross-linked the beta subunits, or formed unstable adducts. Lucifer yellow CH labeled beta specifically without membrane adsorption. Labeling stoichiometries from 1 to 11 mol of lucifer yellow CH per mol of beta were obtained without inhibition of (Na,K)ATPase activity and without significant alteration of the anthroylouabain binding capacity or its association and dissociation kinetics. Anthroylouabain specifically bound to the lucifer-labeled (Na,K)ATPase had a decreased quantum yield, probably due to resonance energy transfer. This suggests that the sites of lucifer attachment on beta are within energy transfer distance from the cardiac glycoside site on alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
J A
JA
Fortes
P A
PA
D000818:Animals; D000873:Anthracenes; D004285:Dogs; D019793:Fluorescein; D005452:Fluoresceins; D005692:Galactose Oxidase; D006023:Glycoproteins; D006834:Hydrazines; D007546:Isoquinolines; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D009439:Neuraminidase; D010042:Ouabain / Q000031:analogs & derivatives; D010084:Oxidation-Reduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors
D000873:Anthracenes; D005452:Fluoresceins; D006023:Glycoproteins; D006834:Hydrazines; D007546:Isoquinolines; D046911:Macromolecular Substances; D010042:Ouabain; C013814:anthroylouabain; C017475:lucifer yellow; D005692:Galactose Oxidase; D009439:Neuraminidase; D000254:Sodium-Potassium-Exchanging ATPase; D019793:Fluorescein