Shift to the Na⁺ from of Na⁺/K⁺‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH₃)₄ATP

Abstract

1. Inactivation of purified Na+/K'-transporting ATPase by the MgATP complex analogue CO(NH~)~ATP, which binds to the low-affinity ATP-binding site, results in the concomitant inhibition of the K+-activated pnitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state.2. Complete inactivation of Na+/K+-transporting ATPase by C O ( N H~)~A T P does not alter the ADP/ATP exchange reaction which is considered to be part of the catalytic activity in the El conformation.3. The enzyme binds eosin at the high-affinity ATP-binding site as measured by the change in eosin fluorescence. Eosin binding to the C~(NH~)~ATP-inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na+ . Inactivation by C O ( N H~)~A T P increased the half-maximal opposing effect of K + on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No eosin fluorescence changes were observed when the Co(NH3)4ATP-inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable complex at the high-affinity ATP-binding site. CrATP thus abolishes eosin binding. It is concluded, that Co(NH3)4ATP interacts with Na+/K+-transporting ATPase in the Ez conformationand arrests it there. This affects eosin binding to the high-affinity ATP-binding site, since the K f sensitivity is lost. A possible interpretation of these differing effects of C O ( N H~)~A T P on partial reactions of N a + / K + -transporting ATPase is that the sodium pump works as an diprotomer. It is likely that the arrest of one ct,P protomer in the E2 conformational state by occupancy of the low-affinity ATP-binding site with C O ( N H~)~A T P induces the Na' form (El form) in the corresponding a,p protomer, as is indicated by the unaffected ADP/ATP exchange and the response of the eosin fluorescence on Na' and K f .It is unclear so far, whether the active transport of Na+ and K + through animal cell membranes is catalyzed by the sodium pump [I, 21 in its M,P protomeric or in its (~( , j j )~ diprotomeric form. Reports on the interaction of N a + / K + -transporting ATPase (Na+/K+-ATPase) with 2',3'-0-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate [3, 41, vanadate [5] as well as the recent report on the incorporation of "Na+ and *'Rb+ into the CrATP-inactivated enzyme and its occlusion in the solubilized form [6] favour the concept that the cr,p protomer might fulfil the minimal requirements of the sodium pump as it is described by the socalled Albers-Post model. There are, however, a number of reports which are not consistent with this assumption (for recent reviews see [7 -91). This sometimes rather indirect data supports the concept of a cooperation of a$ protomers and raises the question how the catalytic c( subunits interact [7 -191. We recently showed that the MgATP complex analogue C O ( N H~)~A T P binds to the low-affinity ATP-binding site, which is found in the Ez conformational state [20, 211. In contrast to...

Modification of the E1ATP binding site of Na+/K+‐ATPase by the chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate blocks the overall reaction but not the partial activities of the E2 conformation

“…As was found with other MgATP complex analogues [lo, 11,[14][15][16], inactivation of Na+/K+-ATPase by Cr(H,O),AdoPP[CH,]P is hindered by the presence of ATP as well. Fig.…”

“…This observation is inconsistent with the classical Albers-Post scheme, in which a single ATP site exists either as E,ATP or as E,ATP site [l]. Yet, the observations are consistent with either the proposal of a monomer combining two ATP sites in one catalytic subunit or the suggestion that membranebound Na+/K+-ATPase may work as an (am, diprotomer with one ATP sitekatalytic a subunit [2][3][4][5][9][10][11][12][13]. In both models, E,ATP and bATP binding sites coexist.…”

Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na+‐exchange reaction of the sodium pump unaltered and shifts its high‐affinity ATP‐binding site to a Na+‐like form

“…This site is most probably identical with the site, to which ATP binds with low-affinity to release the occluded K + during the pump cycle. Modification of the low-affinity ATP-binding site by CO(NH,)~ATP has been shown to lead to a shift towards the Na+ form of the high-affinity ATP-binding site, as was demonstrated recently by measuring eosin fluorescence [17]. We were interested, therefore, to see whether the E; .…”

“…Recently it has been shown that binding of CO(NH,)~ATP to the low-affinity ATP-binding site (in the E2 conformation) shifts the enzyme to the Na' form, when assayed with eosin as a probe for the high affinity site (in the El conformation) [17]. Since C O ( N H~)~P O~ blocks the Na+/K '-transporting ATPase in the E2 conformation in the same way as CO(NH~)~ATP, we tried to determine whether the high-affinity ATP-binding site might similarly be shifted to the N a + form.…”

Renal Physiology

“…The binding, which is competed by ATP, obliterates the Na,K-ATPase activity (overall reaction) and also the K+-phosphatase activity, an E2or low ATP-affinity partial reaction. However, E1 or high ATP-affinity partial reactions, like ATP-ADP exchange, are affected little or not at all (Scheiner-Bobis et al 1989). Such behaviour could be explained (i) if there were two distinct ATP sites per a chain or (ii) if the membranebound enzyme were a functional dimer of the azl protomer, with a single ATP site per a chain.…”