Incorporation of copper into lysyl oxidase.
Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-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 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation.
Kosonen
T
T
Department of Nutrition, University of California, Davis, CA 95616-8669, USA.
Uriu-Hare
J Y
JY
Clegg
M S
MS
Keen
C L
CL
Rucker
R B
RB
D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D020126:Brefeldin A; D003300:Copper / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts; D006031:Glycosylation / Q000187:drug effects; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014415:Tunicamycin / Q000494:pharmacology
D019485:Bone Morphogenetic Proteins; D003517:Cyclopentanes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D014415:Tunicamycin; D020126:Brefeldin A; D003300:Copper; D003513:Cycloheximide; D008249:Protein-Lysine 6-Oxidase; D008666:Metalloendopeptidases; D055395:Bone Morphogenetic Protein 1