STUDIES OF COPPER TRANSPORT IN CULTURED BOVINE CHONDROCYTES

Copper serves as the cofactor for a number of important enzymes in car tilage, as well as in other tissues, including lysyl oxidase, superoxi de dismutase, and cytochrome oxidase. Ceruloplasmin is responsible for the transport of approx. 95% of the copper in serum, but the mechanis ms for intracellular copper transport are unknown. We have demonstrate d recently that a high-molecular-weight cartilage glycoprotein, referr ed to as CMGP, has regions of sequence homology with ceruloplasmin. CM GP also binds copper and has at least some oxidase activity similar to that of ceruroplasmin. Other tissues synthesize intracellular cerulop lasmin-like proteins. The present report represents part of an effort to examine the hypothesis that CMGP is a copper transport protein in c hondrocytes and to characterize the enzymatic activities of CMGP. Thes e studies demonstrate that CMGP is the principal chondrocyte protein l abeled by Cu-67 in vitro and that the label is localized to the mitoch ondria, cytosol, and membrane fractions of sucrose gradients, suggesti ng copper transport through the cell. In parallel experiments, [H-3]le ucine was incorporated into proteins corresponding to the subunits and fragments of CMGP, as described previously, and in a similar distribu tion among the subcellular fractions as labeled copper. Additionally, CMGP has oxidase and ferroxidase activities similar to those of cerulo plasmin.