Elsevier

Life Sciences

Volume 76, Issue 19, 25 March 2005, Pages 2203-2209
Life Sciences

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

https://doi.org/10.1016/j.lfs.2004.10.041Get rights and content

Abstract

This study examined Fe(II)-dependent ATPase activity in OTG (octylthioglucoside) -treated microsomes isolated from Wistar and LEC rats. The ATPase activity of the liver OTG-microsomes from Wistar rats increased sharply in the 5 – 150 μM range of Fe(II) with a K0.5 value of 23.9 ± 3.6 μM, while the activity of LEC rat liver microsomes increased with increasing Fe(II) up to 500 μM with a K0.5 value of 64.4 ± 8.1 μM. The K0.5 values for Fe(II)-dependent ATPase activity of spleen OTG-microsomes were nearly identical at 59.3 μM in the Wistar rat and 63.7 μM in the LEC rats with a similar level of activity at each Fe(II) concentration in both strains of animals. These results indicated that there are two types of Fe(II)-dependent ATPase with different Fe(II) sensitivity, a high sensitive (H) and a low sensitive (L) type, and that the H-type activity was specific to the liver. The H-type activity was, however, deficient in the liver of LEC rats that accumulate copper and iron in hepatocytes as a result of mutations in the Wilson's disease protein (WNDP). On the basis of these results, together with the similarity in optimal conditions required for full activity of the enzyme, we conclude that the Fe(II)-dependent ATPase (H-type) and WNDP may be identical.

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.

Section snippets

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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