Ligands Presumed to Label High Affinity and Low Affinity ATP Binding Sites Do Not Interact in an (αβ)2 Diprotomer in Duck Nasal Gland Na+,K+-ATPase, nor Do the Sites Coexist in Native Enzyme

Abstract

The interaction of ligands deemed to be ATP analogues with renal Na ؉ ,K ؉ -ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2(3)-O-(2,4,6-trinitrophenyl)adenosine 5-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same ␣␤ protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each ␣␤ protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITCbound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITClabeled enzyme, TNP-ADP and ErITC inhibit pnitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC.

Oligomerization of the Na,K-ATPase in Cell Membranes

“…More recently, work with a Na,KATPase preparation from salt-adapted duck nasal gland has supplied evidence for the monomeric ␣␤ heterodimer as the functional unit in the membrane. This preparation, which can be fully phosphorylated, contains primarily monomeric ␣␤ heterodimers (35,36). However, in light of mounting evidence from many methods and systems demonstrating the oligomerization of the Na,K-ATPase, including our current work, it seems likely that in a native membrane environment, the Na,K-ATPase can and does form higher order oligomers.…”

Kinetic characterization of Na,K-ATPase inhibition by Eosin

“…In addition to ATP, the P-type pumps can hydrolyze other phosphate-containing substrates such as para-nitrophenyl phosphate [26,38,39,40], 3-O-methylfluorescein phosphate [41,42], and acetylphosphate [43,44]. We observed that eosin was a potent inhibitor of pNPPase activity (IC 50 = 3.8 ± 0.23 μM; Fig.…”

“…Eosin inhibition of pNPPase may seem surprising, given that FITC-labeled enzyme remains capable of phosphatase activity (40,41). However, previous work has also established that ATP still inhibits pNPPase in FITC treated Na pump.…”

“…One explanation is that during the conformational change from E1 to E2, the tethered FITC molecule is swung out of the active site which allows pNPPase activity [41], but because eosin is not tethered, it is either bound at the site or not attached to the enzyme. An alternative model for the response of the FITC modified pump proposed by Martin and Sachs [40] is that an additional low affinity site for TNP-ADP and erythrosin isothiocyanate appears only after FITC-modification and likely represent a non-physiological allosteric inhibition site. This hypothesis is supported by the dramatically reduced affinities for both TNP-ADP and erythrosin isothiocyanate inhibition of the pNPPase activity in the FITC modified pump compared to the native enzyme [40].…”

“…An alternative model for the response of the FITC modified pump proposed by Martin and Sachs [40] is that an additional low affinity site for TNP-ADP and erythrosin isothiocyanate appears only after FITC-modification and likely represent a non-physiological allosteric inhibition site. This hypothesis is supported by the dramatically reduced affinities for both TNP-ADP and erythrosin isothiocyanate inhibition of the pNPPase activity in the FITC modified pump compared to the native enzyme [40]. Indeed, our finding that eosin inhibits pNPPase in the non-modified enzyme with high affinity is consistent with this hypothesis.…”