INCORPORATION OF COPPER INTO LYSYL OXIDASE

Lysyl oxidase is a copper-dependent enzyme involved in extracellular p rocessing of collagens and elastin., Although it is known that copper is essential for the functional activity of the enzyme, there is littl e information on the incorporation of copper; In the present study we examined the insertion of-copper into lysyl oxidase using Cu-67 in cel l-free transcription/translation. assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post -translational; incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis ( cycloheximide, 10 mu g/ml), glycosylation (tunicamycin, 10 mu g/ml),pr otein secretion (brefeldin A, 10 mu g/ml) and prolysyl oxidase process ing (procollagen C-peptidase inhibitor, 2.5 mu g/ml) together-with 300 mu Ci of carrier-free (CU)-C-67. It was observed that protein synthes is was a prerequisite for copper incorporation, but inhibition of glyc osylation by tunicamycin did not affect the secretion of Cu-67 as lysy l oxidase. Brefeldin A inhibited the secretion of Cu-67-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into ly syl oxidase was not affected. In addition, the inhibition of the extra cellular proteolytic processing of prolysyl oxidase to lysyl oxidase h ad minimal effects on the secretion of protein-bound Cu-67. Our result s indicate that, similar to caeruloplasmin processing [Sate and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolys yl oxidase independently of glycosylation.