Functional Consequences of the Oligomeric Form of the Membrane-Bound Gastric H,K-ATPase
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- 作者：Jai Moo Shin ; Gerhard Grundler ; Jö ; rg Senn-Bilfinger ; Wolfgang Alexander Simon ; and George Sachs
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 13, 2005
- 年：2005
- 卷：44
- 期：49
- 页码：16321 - 16332
- 全文大小：155K
- 年卷期：v.44,no.49(December 13, 2005)
- ISSN：1520-4995
文摘
Cross-linking and two-dimensional crystallization studies have suggested that the membrane-bound gastric H,K-ATPase might be a dimeric 
,
-heterodimer. Effects of an oligomeric structure on thecharacteristics of E1, E2, and phosphoenzyme conformations were examined by measuring bindingstoichiometries of acid-stable phosphorylation (EP) from [
-32P]ATP or 32Pi or of binding of [-32P]ATPand of a K+-competitive imidazonaphthyridine (INT) inhibitor to an enzyme preparation containing ~ 5nmol of ATPase/mg of protein. At <10 
M MgATP, E1[ATP]·Mg·(H+):E2 is formed at a high-affinitysite, and is then converted to E1P·Mg·(H+):E2 and then to E2P·Mg:E1 with luminal proton extrusion.Maximal acid-stable phosphorylation yielded 2.65 nmol/mg of protein. Luminal K+-dependent dephosphorylation returns this conformation to the E1 form. At high MgATP concentrations (>0.1 mM), theoligomer forms E2P·Mg:E1[ATP]·Mg·(H+). The sum of the levels of maximal EP formation and ATPbinding was 5.3 nmol/mg. The maximal amount of [3H]INT bound was 2.6 nmol/mg in the presence ofMgATP, Mg2+, Mg-Pi, or Mg-vanadate with complete inhibition of activity. K+ displaced INT only innigericin-treated vesicles, and thus, INT binds to the luminal surface of the E2 form. INT-bound enzymealso formed 2.6 nmol of EP/mg at high ATP concentrations by formation of E2·Mg·(INT)exo:E1[ATP]·Mg·(H+) which is converted to E2·Mg·(INT)exo:E1P·Mg·(H+)cyto, but this E1P form was K+-insensitive.Binding of the inhibitor fixes half the oligomer in the E2 form with full inhibition of activity, while theother half of the oligomer is able to form E1P only when the inhibitor is bound. It appears that the catalyticsubunits of the oligomer during turnover in intact gastric vesicles are restricted to a reciprocal E1:E2configuration.
,-heterodimer. Effects of an oligomeric structure on thecharacteristics of E1, E2, and phosphoenzyme conformations were examined by measuring bindingstoichiometries of acid-stable phosphorylation (EP) from [-32P]ATP or 32Pi or of binding of [-32P]ATPand of a K+-competitive imidazonaphthyridine (INT) inhibitor to an enzyme preparation containing ~ 5nmol of ATPase/mg of protein. At <10 M MgATP, E1[ATP]·Mg·(H+):E2 is formed at a high-affinitysite, and is then converted to E1P·Mg·(H+):E2 and then to E2P·Mg:E1 with luminal proton extrusion.Maximal acid-stable phosphorylation yielded 2.65 nmol/mg of protein. Luminal K+-dependent dephosphorylation returns this conformation to the E1 form. At high MgATP concentrations (>0.1 mM), theoligomer forms E2P·Mg:E1[ATP]·Mg·(H+). The sum of the levels of maximal EP formation and ATPbinding was 5.3 nmol/mg. The maximal amount of [3H]INT bound was 2.6 nmol/mg in the presence ofMgATP, Mg2+, Mg-Pi, or Mg-vanadate with complete inhibition of activity. K+ displaced INT only innigericin-treated vesicles, and thus, INT binds to the luminal surface of the E2 form. INT-bound enzymealso formed 2.6 nmol of EP/mg at high ATP concentrations by formation of E2·Mg·(INT)exo:E1[ATP]·Mg·(H+) which is converted to E2·Mg·(INT)exo:E1P·Mg·(H+)cyto, but this E1P form was K+-insensitive.Binding of the inhibitor fixes half the oligomer in the E2 form with full inhibition of activity, while theother half of the oligomer is able to form E1P only when the inhibitor is bound. It appears that the catalyticsubunits of the oligomer during turnover in intact gastric vesicles are restricted to a reciprocal E1:E2configuration.