Dissociation and reassociation of prolyl 4-hydroxylase subunits after cross-linking of monomers.
1. Incubation of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with H2O2 leads to a decrease of 50% in the specific activity of enzyme tetramers, followed by dissociation into inactive dimers in which the monomers are covalently cross-linked by S-S bridge formation. 2. Incubation of the enzyme with K3Fe(CN)6 leads to a comparable decrease in activity of enzyme tetramers. Addition of urea leads to dissociation into inactive dimers with similarly cross-linked monomers. 3. Removal of the dissociating agent leads to reassociation of cross-linked dimers to tetramers and to about 50% reactivation. The enzyme is further reactivated by preincubation with dithiothreitol. 4. Dissociation of the enzyme with dithiothreitol, urea or LiCl, or at low pH (4.15) produces inactive monomers, which could not be reassociated.
D000818:Animals; D002642:Chick Embryo; D004229:Dithiothreitol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D008970:Molecular Weight; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects
D006861:Hydrogen Peroxide; D011393:Procollagen-Proline Dioxygenase; D004229:Dithiothreitol