Labeling of the glycoprotein subunit of sodium-potassium ATPase with fluorescent probes

Abstract

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)

Excited-state dynamics of the fluorescent probe Lucifer Yellow in liquid solutions and in heterogeneous media

“…The poor solubility in alcohols might be due to the use of a potassium salt of the ethylenediamine derivative of Lucifer Yellow whereas other groups worked with a lithium salt of the carbohydrazine derivative. 29 Steady-state absorption and fluorescence spectra (Fig. 1) exhibit a single and broad band characteristic of a charge transfer transition and a large Stokes shift, which increases with solvent polarity from about 3600 cm −1 in DMF to about 4300 cm −1 in water.…”

Surface engineering of living myoblasts via selective periodate oxidation

“…However, for in vitro modification of cell surfaces, a far simpler, yet analogous, technique for modifying the surface of living cells exists; the specific oxidation of cell surface vicinal diols, such as those located on sialic acid residues, by mild treatment with sodium periodate (Lenten and Ashwell, 1971). This technique has been routinely used for the detection, characterization and isolation of glycoconjugates (Lee and Fortes, 1985;Roffman et al, 1980;Wilcheck and Bayer, 1987), but has been largely overlooked for the molecular modification of living cell surfaces, except for a small number of studies using blood cells (Fabris et al, 1992;Orr and Rando, 1978;Roffman and Wilcheck, 1986;Tolvanen and Gahmberg, 1986). Since most mammalian cells are grown in adherent cultures and are thus affected by cell-cell and cell-substrate interactions, we aimed to investigate the effectiveness of sodium periodate for the molecular engineering of adherent cell lines and to compare this technique with metabolic ketone incorporation using ManLev.…”

The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site

“…In this study we have used several fluorescent probes bound to the enzyme to obtain additional information about the structural and dynamic properties of the oligosaccharides of /3 and af3 interactions. We have used the procedures of Lee and Fortes (1985) to label the carbohydrates with the fluorophore lucifer yellow (LY). We have analyzed the steady-state and dynamic fluorescence properties of bound LY to deduce the probe's environment and motional properties.…”