Abstract
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 μg/ml), glycosylation (tunicamycin, 10 μg/ml), protein secretion (brefeldin A, 10 μg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 μg/ml) together with 300 μCi 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 67Cu-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, copper is inserted into prolysyl oxidase independently of glycosylation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 283-289 |
| Number of pages | 7 |
| Journal | Biochemical Journal |
| Volume | 327 |
| Issue number | 1 |
| State | Published - 1997 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Incorporation of copper into lysyl oxidase. / Kosonen, T.; Uriu-Hare, J. Y.; Clegg, M. S.; Keen, Carl L; Rucker, R. B.
In: Biochemical Journal, Vol. 327, No. 1, 1997, p. 283-289.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Incorporation of copper into lysyl oxidase
AU - Kosonen, T.
AU - Uriu-Hare, J. Y.
AU - Clegg, M. S.
AU - Keen, Carl L
AU - Rucker, R. B.
PY - 1997
Y1 - 1997
N2 - 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 μg/ml), glycosylation (tunicamycin, 10 μg/ml), protein secretion (brefeldin A, 10 μg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 μg/ml) together with 300 μCi 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 67Cu-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, copper is inserted into prolysyl oxidase independently of glycosylation.
AB - 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 μg/ml), glycosylation (tunicamycin, 10 μg/ml), protein secretion (brefeldin A, 10 μg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 μg/ml) together with 300 μCi 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 67Cu-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, copper is inserted into prolysyl oxidase independently of glycosylation.
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UR - http://www.scopus.com/inward/citedby.url?scp=0030713424&partnerID=8YFLogxK
M3 - Article
C2 - 9355764
AN - SCOPUS:0030713424
VL - 327
SP - 283
EP - 289
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 1
ER -