ATP Hydrolytic Activity of an Iron-Stimulated P-type ATPase of Mouse Liver Microsomes

Fe(II)/Cu(I)-dependent P-type ATPase activity in the liver of long-evans cinnamon rats

“…The ATPase activity of OTGmicrosomes from Wistar rats increased sharply in the 5-150 AM range of Fe(II) and then declined, with a mean (F SE) K 0.5 of 23.9 F 3.6 AM (n = 3). These characteristics are essentially the same as those we reported previously for Fe(II)-dependent ATPase in ddy mice liver OTG-microsomes (Takeda et al, 2000). In contrast, ATPase activity of LEC rat liver OTG-microsomes rose with increasing Fe(II) concentrations up to 500 AM with a mean (F SE) K 0.5 value of 64.4 F 8.1 AM (n = 3).…”

“…1. An acidic pH and a high salt concentration are required for maximum Fe(II)-dependent ATPase activity (Takeda et al, 2000). Under the optimal conditions of pH5.0 and 300mM KCl, maximum activities of approximately 0.75 Amol Pi/mg/h for Wistar rats and approximately 0.5 Amol Pi/mg/h for LEC rats were reached at 150 AM Fe(II) and 500 AM Fe(II), respectively.…”

“…Since trace amounts of copper present in the preparation medium for microsomes are sufficient to fully activate Cu(I)-dependent ATPase in the liver (Takeda et al, 2000), the OTG-microsomes were pre-treated with 1 mM bathocuproine disulfonate (BCS), a copper chelator, and then washed twice using centrifugation. The Cu(I) concentration dependency of the ATPase of BCS-treated OTG-microsomes from the liver of Wistar and LEC rats is shown in Fig.…”

“…The Fe(II)-dependent ATPase activity was assayed in triplicate at 378C in 100 Al of 50 mM acetate buffer at pH5.0 containing 300 mM KCl, 3 mM MgSO 4 , 10 mM DTT, 3 mM ATP and OTG-microsomes (0.4 mg/ml) at varying Fe(II) concentrations up to 500 AM (Takeda et al, 2000). The stock solution containing acetate buffer, KCl, MgSO 4 , DTT and FeCl 3 was made at least a week before use in order to ensure reduction of Fe(III) to Fe(II).…”

“…In previous papers, we characterized a Fe(II)/Cu(I)-dependent ATPase in mouse liver microsomes treated with octylthioglucoside (OTG) (Takeda et al, 1999(Takeda et al, , 2000. We showed this ATPase belonged to the P-type ATPase family as it was inhibited by vanadate, a specific inhibitor of the P-type ATPase, and was phosphorylated by ATP in a hydroxylamine-sensitive manner.…”