Isolation and characterization of Cox17p from porcine heart by determining its survival-promoting activity in NIH3T3 cells.
We have found that the gel filtration fraction of porcine heart extract clearly promoted the survival of NIH3T3 fibroblast cells in the serum-free medium condition. A structural analysis showed that the active fraction contained a novel peptide, porcine Cox17p (p-Cox17p), which was recently reported by Chen et al. as dopuin (Z. W. Chen et al., Eur. J. Biochem. 249 (1997) 518-522). Porcine Cox17p/dopuin possesses high sequence homology to the product of human COX17 gene (h-Cox17p). Although Cox17p has been implied to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome oxidase in yeast, its role in mammalian cells is unknown. In this study, we chemically synthesized p-Cox17p to investigate its biological effects. Refolding experiments of synthesized linear p-Cox17p revealed the existence of mostly one pattern of three intrachain disulfide bridges similar to that of native p-Cox17p, because the main oxidized p-Cox17p was completely co-eluted with the natural product. The addition of heavy metal ions such as copper, zinc and cadmium significantly inhibited the formation of the oxidized form, suggesting that reduced p-Cox17p may interact directly with these metal ions. The reduced and oxidized forms of p-Cox17p were also confirmed to promote the survival of NIH3T3 cells in serum-free medium as observed with the natural product, indicating that Cox17p may be a bioactive peptide.
Takenouchi
T
T
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Fujimoto
M
M
Shimamoto
A
A
Munekata
E
E
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D000079922:Copper Transport Proteins; D016895:Culture Media, Serum-Free; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D010084:Oxidation-Reduction; D017510:Protein Folding; D011506:Proteins / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014020:Tissue Extracts / Q000737:chemistry
C100359:COX17 protein, S cerevisiae; C491006:COX17 protein, human; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; C491005:Cox17 protein, mouse; D016895:Culture Media, Serum-Free; D018832:Molecular Chaperones; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014020:Tissue Extracts