Fe(II)/Cu(I)-dependent P-type ATPase activity in the liver of long-evans cinnamon rats
Introduction
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.
More than 200 members of the P-type ATPase family have been identified. Based on sequence analysis, cation specificity and membrane topology, the P-type ATPases are divided into 5 subfamilies (Axelsen and Palmgren, 1998), with the heavy metal transporting ATPase belonging to the type IB P-type ATPase subfamily. The first mammalian copper-transporting ATPases were identified as a result of searches for the genes involved in disorders of copper metabolism such as Menkes disease and Wilson's disease (Vulpe et al., 1993, Tanzi et al., 1993, Bull et al., 1993, Yamaguchi et al., 1993). Wilson's disease is caused by a mutation in the ATP7B gene that results in impaired intracellular hepatocyte copper transport leading to accumulation of copper in the cytosol. Although the Wilson's disease protein (WNDP) is dependent on ATP to transport copper and forms a phosphorylated intermediate in a manner similar to other P-type ATPases (Hung et al., 1997, Payne and Gitlin, 1998, Voskoboinik et al., 2001), the ATP hydrolytic properties of the protein remain poorly understood.
The Long-Evans with a cinnamon-like coat color (LEC) rat develops hepatitis spontaneously as a result of abnormal copper accumulation in the liver (Li et al., 1991). There is evidence that expression of the rat homologue of the Wilson's disease gene is entirely absent from tissues in the LEC rat (Wu et al., 1994, Yamaguchi et al., 1994), indicating that this strain of rat is a bona fide animal model of Wilson's disease and therefore important for studying liver pathophysiology and pathways of copper transport.
As the LEC rat develops both abnormal hepatic iron and copper accumulation (Kato et al., 1993), we considered it would be interesting to investigate Fe(II)/Cu(I)-dependent ATPase activity in these animals. If it could be demonstrated that LEC rats lacked Fe(II)/Cu(I)-dependent ATPase activity, this would imply that ATPase and the WNDP protein are identical. The present study examined mainly Fe(II)-dependent rather than Cu(I)-dependent ATPase activity in the liver and spleen microsomes of LEC rats because of the simplicity of assay of the former enzyme. We found two types of Fe(II)-dependent ATPase activities with different Fe(II) sensitivity in the liver of control Wistar rats, but were unable to detect Fe(II)-dependent ATPase with high Fe(II) sensitivity in the liver of LEC rats.
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Materials
Bathophenanthroline disulfonate (BPS), bathocuproine disulfonate (BCS) and octylthioglucoside (OTG) were obtained from Dojindo Laboratories (Kumamoto, Japan). The Wistar and LEC rats were purchased from KBT Oriental (Fukuoka, Japan).
Membrane preparations
Adult male rats were anaesthetized and then sacrificed by decapitation. The liver and spleen were then isolated and homogenized using a Daunce homogenizer (loose fitting), in 0.25 M sucrose containing 1 mM EGTA and 5 mM HEPES at pH7.4. Following two centrifugations
ATPase assay
The Fe(II)-dependent ATPase activity was assayed in triplicate at 37°C in 100 μl of 50 mM acetate buffer at pH5.0 containing 300 mM KCl, 3 mM MgSO4, 10 mM DTT, 3 mM ATP and OTG-microsomes (0.4 mg/ml) at varying Fe(II) concentrations up to 500 μM (Takeda et al., 2000). The stock solution containing acetate buffer, KCl, MgSO4, DTT and FeCl3 was made at least a week before use in order to ensure reduction of Fe(III) to Fe(II). Following incubation at 37°C for 15 min, the reaction was started by
Results
The ATPase activity of OTG-microsomes from the liver of Wistar and LEC rats was examined as a function of Fe(II) concentration and the results are shown in Fig. 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 μmol Pi/mg/h for Wistar rats and approximately 0.5 μmol Pi/mg/h for LEC rats were reached at 150 μM Fe(II) and 500 μM
Discussion
During the course of isolation of the WNDP protein, we detected Fe(II)/Cu(I)-dependent ATPase activity in OTG-treated microsomes from mouse liver (Takeda et al., 2000). However, as we were unable to purify or sequence the ATPase enzyme, it remained uncertain whether or not the ATPase activity was attributable to the WNDP protein. Recently, an acylphosphate intermediate formed during the hydrolysis of ATP by the WNDP protein has been characterized (Tsivkovskii et al., 2002). For optimal
Acknowledgements
This work was supported in part by a Special Grant from the Ministry of Labor for Occupational Health Studies.
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