F. Cornelius, PHOSPHORYLATION DEPHOSPHORYLATION OF RECONSTITUTED SHARK NA-ATPASE - ONE PHOSPHORYLATION SITE PER ALPHA-BETA PROTOMER(,K+), Biochimica et biophysica acta. Biomembranes, 1235(2), 1995, pp. 197-204
In the present investigation reconstitution of Na+,K+-ATPase increases
the number of phosphorylation sites (EP) of solubilized enzyme from 4
.2 +/- 0.3 nmol/mg to 6.9 +/- 0.6 nmol/mg. The latter figure correspon
ds to one phosphorylation site per alpha beta-protomer. A cholesterol
content > 10 mol% in the liposome bilayer and a high extracellular [Na
+] are necessary to obtain this high value. Spontaneous dephosphorylat
ion after maximum phosphorylation in Na+ is biphasic both in solubiliz
ed enzyme and after reconstitution. The rate of dephosphorylation comp
ares with the specific hydrolytic Na+-ATPase activity measured at exac
tly identical conditions for all three preparations assuming parallel
dephosphorylation of at least two phosphointermediates. The distributi
on of EP-species is found to vary among the three enzyme preparation u
sed, i.e., membrane bound, solubilized, and reconstituted Na+,K+-ATPas
e, however in all the equilibrium is strongly poised away from the E(1
)P-form.