In Vitro and In Vivo Studies on the Degradation of Metallothionein

Degradation of metallothionein (MT) from rat liver was examined. Degradation of apo-MT by liver homogenate was greater than that by cytosol. At pH 5.5, degradation by homogenate was more than that at pH 7.2. These findings suggest that proteases that function at acidic pH are probably involved in MT degradation. Because lysosomes are the principal subcellular organelles that contain acid proteases (cathepsins), we compared the degradation of apo-MT by lysosomes and cytsosol. Apo-MT was degraded about 400 times faster by lysosomal fraction than by cytosolic fraction. To determine the relative importance of different cathepsins, we used different inhibitors. Leupeptin, which inhibits cathepsins B and L, inhibited the degradation of apo-MT by 80%, implying that cathepsins B and/or L might be very important in the intracellular turnover of MT. Cathepsin D appeared to be the least significant, because apo-MT degradation was reduced by about 20% by inhibiting cathepsin D. When we extended this study with purified cathepsins, we obtained the same answer, i.e., the ability of different cathepsins to degrade apo-MT was in the following order: cathepsin B >> cathepsin C > cathepsin D. While apo-MT was susceptible to degradation, ZnMT and CdMT were highly resistant to degradation. Coincubation of ZnMT or CdMT with either lysosomal extract or purified cathepsins did not result in any appreciable degradation even after 16 hr. However, longer incubations did result in some degradation, especially by purified cathepsin B. Interestingly, CdMT degraded little faster than ZnMT by both lysosomal extract as well as purified cathepsin B. These data suggest that metals protect MT from rapid proteolysis. By metal-titration study, we found that at least 5 moles of Zn equivalent/mole of MT dramatically reduced the degradation, by both lysosomal fraction and purified cathepsin B. This indicates that metal release might be a prerequisite for the initiation of degradation. Release of metals is possible in the lysosomes because the intralysosomal pH is close to 5.0. Therefore, we determined the displacement of metals (Zn) as a function of pH. We found that at a pH of 4.5, nearly 70% Zn was removed, and at pH 4.0 nearly all Zn was displaced from MT. We propose, therefore, that lysosomes are probably important in the in vivo degradation of MT, and that metal release is a prerequisite for degradation. With the release of metals in the acidic pH of lysosomes, MT becomes more susceptible t