Establishment of a method of analyzing ATP7B function.

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Wilson disease is a genetic disorder of copper metabolism caused by a defect in a copper-transporting P type ATPase (ATP7B). Gene analysis has been used for the diagnosis of Wilson disease. Analysis of ATP7B function will also be useful for diagnosis of this disease. However, no method for analyzing of ATP7B function has been reported. We investigated to establish a method for analyzing of ATP7B function. At first we studied about ATP7A function with cultured fibroblasts from patients with Menkes disease that is caused by another copper-transporting ATPase (ATP7A).【Materials and Methods】 Cultured fibroblasts from patients with Menkes disease, patients with Wilson disease and control. Harvested cells were homogenized and centrifuged. The supernatants were used. A glutathione-Cu solution, ATP solution were added to the samples, and mixed. After then, the mixture was added to Luciferase reagent, and then the amount of ATP concentration in the mixture was measured using an Auto Limat.【Results and Discussion】The activity of ATP7A/B in the control cells was 10-200pmol/mg.pro./min, showing that the level of normal control is large wide. In the cells from patients with Menkes disease, the activity levels were also large wide ; in some patients, the activity was lower than the normal ranges, but in some of them, the level was within the normal range. We concluded that this method is not available to measure the activity of ATP7A/B function.