Role of hepatic lysosomes in the degradation of metallothionein.
The degradation of metallothionein (MT) by rat liver was examined. Degradation of MT by liver homogenate was greater than by cytosol. In addition, MT degradation by the homogenate at pH 5.5 was more than that at pH 7.2. Because lysosomal proteases function at acidic pH, these findings suggest the importance of lysosomes in MT degradation. The degradation by the lysosomal fraction was about 400-fold greater than that by the cytosol. Because cathepsins are the principal lysosomal proteases, we used cathepsin-specific inhibitors, such as leupeptin, E-64 and pepstatin, to determine the relative importance of different cathepsins in degrading MT. The study reveals that cathepsin B and/or L is (are) probably the most important enzyme(s) in degrading hepatic MT, because leupeptin, which blocks cathepsin B and L activity, inhibited the degradation of apo-MT by about 80%. Cathepsin D appears to be of least importance in MT degradation, because inhibition of this enzyme by pepstatin reduced degradation by only 20%. Studies on the degradation of apo-MT, ZnMT, and CdMT indicated that apo-MT is about 1500-fold more sensitive to degradation than ZnMT and CdMT. These data suggest that metals protect MT from degradation. This is further supported by a reconstitution experiment, which shows that with a progressive decrease of MT: metal ratio following titration of apo-MT by metals, there is a concomitant reduction in degradation. At a lysosomal pH of around 4.7, about 60% of Zn and 20% of Cd are displaced from MT, thereby making it susceptible to degradation. We propose, therefore, that lysosomes are probably important for MT degradation in vivo and that metal release is a prerequisite for degradation. With the release of metals, MT becomes susceptible to degradation, which is probably accomplished by the lysosomal cathepsins, in particular cathepsins B and L.
Choudhuri
S
S
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City 66160-7417.
McKim
J M
JM
Klaassen
C D
CD
D000818:Animals; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008668:Metallothionein / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D007976:Leupeptins; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D008668:Metallothionein; D002403:Cathepsins; D007930:Leucine; C032854:leupeptin; C024974:E 64; C031375:pepstatin