Degradation of zinc-metallothionein in monolayer cultures of rat hepatocytes.
The degradation of zinc-metallothionein (MT) was studied in monolayer cultures of adult rat hepatocytes. Hepatocytes were incubated overnight in serum-free medium containing either [35S]cysteine or [3H]leucine and 100 microM zinc to induce MT synthesis. Total cellular 35S-MT was measured in the heat-stable extract of cell homogenate and quantified by fast protein liquid chromatography. When zinc was removed from the medium, 35S-MT turnover was almost 3-fold faster than that of [3H]Leu protein (t1/2 = 11 and 29 hr, respectively). The decrease in the cellular level of 35S-MT reflected degradation since less than 1% of total cellular 35S-MT was secreted into the medium. The rate of MT degradation was inversely proportional to cellular zinc content. In contrast, the degradation of [3H]Leu protein was not affected by changes in cellular zinc concentration. Chloroquine, a lysosomotrophic amine, and tosyl lysine chloromethyl ketone, an inhibitor of trypsin-like neutral protease activity, inhibited 35S-MT degradation by 65% and 50%, respectively, when cells were incubated in medium with 1 microM zinc. Turnover of [3H]Leu protein, but not 35S-MT, was enhanced by insulin deprivation. These data suggest that the degradation of hepatic MT (i) is primarily regulated by cellular zinc content and (ii) occurs in both lysosomal and nonlysosomal compartments.
Chen
M L
ML
Vitamin and Mineral Nutrition Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705.
Failla
M L
ML
D000818:Animals; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D003470:Culture Media; D008099:Liver / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008668:Metallothionein / Q000378:metabolism*; D051381:Rats; D015032:Zinc / Q000378:metabolism*
D003470:Culture Media; D008668:Metallothionein; D015032:Zinc