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77 d and particularly of the animals with systemic jaundice aged 86 d. These animals showed also clearly increased copper levels in the lysosome-specific fractions no. 9. The relatively high concentration of copper in fraction no. 10 derives from cytosolic contamination. Rats treated with DPA from d 64 to 107 showed only marginally elevated copper levels in the density fractions. The concentrations of MT in the fractions (Fig. 3B) paralleled those of copper. Accordingly, treatment of 77-d-old rats with DPA significantly decreased the MT levels in the high-density fraction no. 1 by 75%. MT in this fraction, however, was insoluble and could be analyzed only after solubilization with guanidinium thiocyanate and 2-mercaptoethanol.

Ultrastructurally, the insoluble material consisted of lysosomes containing electron-dense granules (Fig. 4A). Using energy-dispersive X-ray microanalysis, the electron-dense material could be attributed to copper (Fig. 4B).

High-performance liquid chromatography (HPLC) analysis of the solubilized material revealed a copper-containing component of 7.3 kDa, corresponding to 50% of the apparent molecular weight of native MT (Fig. 5). This component crossreacted with MT-specific antibodies in an enzyme-linked immunosorbent assay (ELISA) (not shown). A similar material, although at lower concentration, was isolated in addition to native MT, also from the lysosome-specific fraction no. 9 of the density gradient (Fig. 5).

The solubilization of the MT polymers by DPA was investigated in vitro. In the presence of 185 mM DPA, both copper and MT were solubilized almost totally within 5 min (results not shown). After HPLC analysis, copper was recovered at a relative elution volume of 2.0, corresponding to an apparent molecular weight of 6.6 kDa (Fig. 6). This fraction was bluish purple with an absorption maximum at 520 nm, which has been reported to be specific for thiol-copper(II) complexes (20). In contrast, copper in the cytosol was virtually not mobilized by DPA (Fig. 6).

Fig. 1. Transmission electron micrographs from the livers of LEC rats aged 77 d (A-C), aged 87 d with systemic jaundice (D), and aged 84 d after treatment with d-penicillamine from d 77 (E, F). Original magnification: A, B, E: 2.650X, C: 5.000X, D: 1.670X, F: 4.320X. Arrows indicate the localization of electron-dense particles. [From Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

Fig. 1. Transmission electron micrographs from the livers of LEC rats aged 77 d (A-C), aged 87 d with systemic jaundice (D), and aged 84 d after treatment with d-penicillamine from d 77 (E, F). Original magnification: A, B, E: 2.650X, C: 5.000X, D: 1.670X, F: 4.320X. Arrows indicate the localization of electron-dense particles. [From Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

Fig. 2. Total and noncytosolic copper contents in the liver of LEC rats with and without treatment with d-penicillamine (DPA) (mean ± SD, n = 3-11). 'Significantly different from LEC rats, age 62 d (p < 0.05); "significantly different from LEC rats, age 77 d (p<0.05). [From Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

Fig. 2. Total and noncytosolic copper contents in the liver of LEC rats with and without treatment with d-penicillamine (DPA) (mean ± SD, n = 3-11). 'Significantly different from LEC rats, age 62 d (p < 0.05); "significantly different from LEC rats, age 77 d (p<0.05). [From Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

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