Alphabeta protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: formation of higher oligomers does not modify molecular activity

Abstract

The distance that separates alphabeta protomers of the Na(+), K(+)-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of alphabeta protomers and either N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 A farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism.

Figure 1

Electron micrograph of purified Na⁺,K⁺-ATPase preparation. A Na⁺,K⁺-ATPase preparation suspended in Tris, pH 7.5 buffer was adhered to a formvar-coated copper grid, negatively stained with 1% uranyl acetate and viewed under an 80 KV beam at ×200,000 magnification. Na⁺,K⁺-ATPase molecules in this negatively stained preparation appear as white dots. (Scale bar = 0.1 μm.)

Figure 2

Model of the interaction of Na⁺ pumps and cytoskeleton in different membrane preparations.

Figure 3

Effects of varied concentrations of ouabain and ouabain derivatives on PNPPase activity. Purified Na⁺,K⁺-ATPase was incubated at 20 μg/ml with varied concentrations of ouabain, AO, FO, and NBDO for 30 min at room temperature, and the PNPPase activity was measured as described in Methods. The data were fitted to a sigmoidal dose-response curve by using prizm 2.01 (GraphPad, San Diego). The fitted parameters and symbols are: ○ = ouabain, EC₅₀ = 6.4 nM; ● = AO, EC₅₀ = 64 nM; □ = NBDO, EC₅₀ = 210 nM; ■ = FO, EC₅₀ = 860 nM.