CHARACTERIZATION OF INTRACELLULAR COPPER POOLS IN RAT HEPATOCYTES USING THE CHELATOR DIAMSAR

When hepatocytes are incubated with the chelator diamsar, two pools ca n be identified, which we have termed extractable and nonextractable. On entering the hepatocyte, Cu-67 first associates with the extractabl e pool and, after similar to 2 h, moves to the nonextractable pool. Bo th pools demonstrate saturation and are filled as a function of Cu con centration and incubation time. Using the Michaelis-Menten equation, w e have estimated the size of the pools after incubation with Cu-67 for 30 min and 4 h. During this period the extractable pool decreases in size from 200 +/- 27 to 116 +/- 5 pmol/mu g DNA, whereas the nonextrac table pool increases from 28 +/- 9 to 77 +/- 11 pmol/mu g DNA. Movemen t of Cu from the nonextractable pool to the extractable pool is slow a nd incomplete. Using [H-3]diamsar, we demonstrate that uptake of the c helator is not rate limiting and probably does not occur by pinocytosi s. Incubation with diamsar does not affect the activity of superoxide dismutase or cytochrome-e oxidase, although it does prevent the incorp oration of Cu-67 into ceruloplasmin. Incubation with zinc, which induc es metallothionein, results in an increase in Cu-67 associated with th e nonextractable pool, suggesting that Cu-67-metallothionein constitut es at least part of the nonextractable pool.