Preparation of Na<sup>+</sup>,K<sup>+</sup>-ATPase with Near Maximal Specific Activity and Phosphorylation Capacity:  Evidence That the Reaction Mechanism Involves All of the Sites

Because nearly all structure/function studies on Na ؉ / K ؉ -ATPase have been done on enzymes prepared in the presence of SDS, we have studied previously unrecognized consequences of SDS interaction with the enzyme. When the purified membrane-bound kidney enzyme was solubilized with SDS or TDS concentrations just sufficient to cause complete solubilization, but not at concentrations severalfold higher, the enzyme retained quaternary structure, exhibiting ␣,␣-, ␣,␤-, ␤,␤-, and ␣,␥-associations as detected by chemical cross-linking. The presence of solubilized oligomers was confirmed by sucrose density gradient centrifugation. This solubilized enzyme had no ATPase activity and was not phosphorylated by ATP, but it retained the ability to occlude Rb ؉ and Na ؉ . This, and comparison of cross-linking patterns obtained with different reagents, suggested that the transmembrane domains of the enzyme are more resistant to SDSinduced unfolding than its other domains. These findings (a) indicate that the partially unfolded oligomer(s) retaining partial function is the intermediate in the SDS-induced denaturation of the native membrane enzyme having the minimum oligomeric structure of (␣,␤,␥) 2 and (b) suggest potential functions for Na ؉ /K ؉ -ATPase with intrinsically unfolded domains. Mixtures of solubilized/ partially unfolded enzyme and membrane-bound enzyme exhibited cross-linking patterns and Na ؉ occlusion capacities different from those of either enzyme species, suggesting that the two interact. Formation of the partially unfolded enzyme during standard purification procedure for the preparation of the membrane-bound enzyme was shown, indicating that it is necessary to ensure the separation of the partially unfolded enzyme from the membrane-bound enzyme to avoid the distortion of the properties of the latter.

Section: Table Imentioning

confidence: 60%

mentioning

confidence: 99%

Interaction of SDS with Na+/K+-ATPase

Ivanov

Gable

Askari

2004

“…Each mutation in the conserved GDASE sequence induced different affinity changes in the two different ATP effects and the affinity for pNPP providing further support for the presence of a single ATP binding site/␣-chain as proposed (12,37,43). Quite recently, three interesting studies appeared, the association of ␤Ϫ␤ chain (51) and the association of expressed ␣-chains by MgATP (54) and the specific activity of the Na/K-ATPase activity of the tetraprotomeric Na/K-ATPase fraction was approximately half that of the diprotomeric and protomeric fractions, using a solubilized dog kidney enzyme (21), which nicely explains both that protomer is sufficient for Na/K-ATPase activity (46,48) and oligomericity is required for the enzyme (11-13, 19 -20, 37, 43, 51-52, 54). These data are consistent with a hypothesis in which each ␣-subunit contains 1 mol of a high affinity ATP binding site for EP formation and 1 mol of low affinity ATP binding site for EATP formation, in the 2n-mer, not only in Na/K-ATPase but also in H/K-ATPase, possibly as (EP:EATP) 2 (21, 37, 43, 52).…”

Section: Larger Apparent Affinity Decrease For Atp Effects Of Each Mumentioning

confidence: 77%

“…Unfortunately it was not possible to measure the amount of EATP during turn over in the present enzyme preparations used which were expressed in HeLa cells, because of their purity, namely Ͻ ϳ10 pmol of EP/mg of protein. If one takes the amount of EP ϩ EATP (37,43) or the amount of bound ouabain (48) or phosphoenzyme from P i in the presence of ouabain (46) as the amount of active site, the turnover number should decrease by half (44 -46). Although the value of V max is semi-quantitative, the changes in the relative values obtained in the same series of experiments were compared, and these had no effect on our present conclusion.…”

Section: Larger Apparent Affinity Decrease For Atp Effects Of Each Mumentioning

confidence: 99%

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Imagawa

Kaya

Taniguchi

2003

.5 ) and the apparent K m for Mg 2؉ , Na ؉ , K ؉ , and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was ϳ2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20ϳ50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, K m l /K m h , for each mutant was 1.3ϳ3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each K m P value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442 GDASE 446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg 2؉ , Na ؉ , and K ؉ effects.

“…We recently perfected a method for preparing Na ϩ ,K ϩ -ATPase from duck nasal glands that reliably yields enzyme of maximal theoretical phosphorylation capacity (9). We have used this preparation to reevaluate two studies relevant to the issue of whether or not protomer interaction is involved in the manifestation of putative high and low affinity ATP binding sites.…”

Section: Namentioning

confidence: 99%

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

Martin

Sachs

2000

Self Cite

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.