An Extra Phosphorylation of Na+, K+-ATPase by Paranitrophenylphosphate (pNPP): Evidence for the Oligomeric Nature of the Enzyme1

Abstract

Paranitrophenylphosphate (pNPP) induced fluorescence changes in fluorescence isothiocyanate (FITC)-labeled Na+,K(+)-ATPase preparations. The extents of changes were similar to those induced by acetylphosphate (AcP) accompanying accumulation of a K(+)-sensitive phosphoenzyme (E2P) and an ouabain bound phosphoenzyme in the presence of Mg2+ and 16 mM Na+. Phosphoenzymes formed from [32P]pNPP were shown to turn over. The ratio of the maximum amount of the phosphoenzyme formed from pNPP to that of the phosphoenzyme formed from ATP and that of the ouabain-enzyme complex under steady-state conditions was shown to be close to 1:0.5:1. Such extra phosphorylation has hitherto only been observed in a transient state with the additions of high concentrations of ATP [Peluffo, R.D., Garrahan, P.J., and Rega, A. (1992) J. Biol. Chem. 267, 6596-6601]. Our data are compatible with the simultaneous presence of high and low affinity ATP-binding sites in Na+,K(+)-ATPase [Hamer, E. and Schoner, W. (1993) Eur. J. Biochem. 213, 743-748]. The maximum amount of paranitrophenol-sensitive fraction to synthesize [32P]pNPP in fully accumulated ADP-sensitive phosphoenzyme (E1P) from [32P]ATP was around 1/4 of the amount of ouabain-enzyme complex. These data and others indicate that a much higher degree of oligomerization, rather than (alpha beta)2, may be the functional unit of the enzyme in the membranes.

Half-site Modification of Lys-480 of the Na+,K+-ATPase α-Chain with Pyridoxal 5′-Diphospho-5′-adenosine Reduces ATP-dependent Phosphorylation Stoichiometry from Half to a Quarter

“…27) to that of ouabain-enzyme complex. Present data are consistent with our previous data, which showed that the ratio of the maximum amount of ouabain-enzyme complex to that of phosphoenzyme was ϳ2 and was independent of the specific activity of the enzyme preparations from dog kidney purified with SDS (2000 -2750 mol of P i /mg of protein/h) or from pig kidney purified with sodium deoxycholate-NaI (500 -1200 mol of P i /mg of protein/h) (27). It has been reported that pig kidney Na ϩ ,K ϩ -ATPase preparations contained ϳ10% heterogeneity (38).…”

“…The data showed that AP 2 PL modification and the AP 2 PL-FITC modification reduced the amount of phosphoenzymes from ATP to 50 and 6%, respectively, indicating that ϳ0.5 mol of AP 2 PL probe binding at Lys-480 and ϳ0.9 mol of FITC probe binding/␣-chain reduced the value (in moles of ATP-dependent phosphorylation/mol of ␣-chain) from 0.5 to 0.25 and 0.03 (Fig. 1B), with little influence on the half-site phosphorylation by AcP (Table I) and full-site ouabain binding in the presence of Mg 2ϩ and P i as reported previously (27). Stopped-flow measurements showed that the addition of 10 M ATP to the AP 2 PL enzyme and AP 2 PL-FITC enzyme preparations, respectively, in the presence of 4 mM Mg 2ϩ and 16 mM Na ϩ increased the AP 2 PL fluorescence intensity to ϳ3% despite 50 and 94% reduction in phosphorylation by ATP (Table I).…”

“…The amino acid sequence analysis of the ␣-chains of pig kidney enzymes showed that nearly all the sequence started with Gly-1, which also excluded proteolytic breakdown of the ␣-chain to produce 50% heterogeneity (not shown). The present data showed half-site phosphorylation by ATP and AcP, respectively, and half-site reactivity to AP 2 PL (a quarter-site phosphorylation by ATP after the half-site modification by AP 2 PL), while they showed full-site ouabain binding capacity from P i with Mg 2ϩ (27), ATP-sensitive full-site reactivity toward FITC and nearly full-site ATP binding capacity, respectively (20). From these data it can be concluded that Mg 2ϩ -and Na ϩ -dependent phosphorylation from ATP or AcP under steady state conditions occurred on only half of the Asp-369 residues in the ␣-chains of the membrane-bound Na ϩ ,K ϩ -ATPase.…”

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.…”

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

“…Abbreviations: Cr(H20)4Adoi , i > [CH 2 ]i', ß,y bidentate complex of chromium(III)-tetraaqua-adenylyl [ß,y-methylene] diphosphonate; CofNHs^ATP, ß,y bidentate complex of cobalt(III)-tetramino-adenosine-5'-triphosphate; Co(NH3)4P04, tetramine cobalt(III)phosphate; FITC, fluorescein 5'isothiocyanate; NBD-C1, 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole, 7-chloro-4-nitro-benzofurazane; EiATP site, nucleotide-binding site of Na + /K + -ATPase with high affinity for ATP; E2ATP site, nucleotide-binding site of Na + /K + -ATPase with low affinity for ATP phenomena of superphosphorylation from [y-32 P]ATP [4], double labeling with ATP analogs [5] or double phosphorylation from para-nitrophenylphosphate [6]. All these observations, including the observation of a positive cooperative effect of 2'-ODANS-8-N 3 -ATP in the inactivation of Na+/K+-ATPase [7] led to the conclusion that two simultaneously existing ATP sites need to cooperate during ATP-driven Na+/ K + transport [7,8].…”