title: Reductive cleavage and reformation of the interchain and intrachain disulfide bonds in the globular hexameric domain NC1 involved in network assembly of basement membrane collagen (type IV).
abstract: The formation of collagen IV dimers in the extracellular space requires the association of two C-terminal globular domains giving rise to a large hexameric structure NC1 (Mr = 170,000). NC1 hexamer was purified from collagenase digests of a mouse tumor and several human tissues. It was shown by electrophoresis to consist of two kinds of cross-linked, dimeric segments, Da and Db (Mr about 50,000), and monomeric segments in a molar ratio of about 3:1. In the native hexamers free SH groups were detectable by N-[14C]ethylmaleimide and other sulfhydryl reagents. They account for 4-11% of the total number of cysteine residues with some variations between preparations from different sources and in the distribution between monomers and dimers. Reduction with 10 mM dithioerythritol under non-denaturing condition completely converted dimers into monomers and allowed the alkylation of all twelve cysteine residues present in each monomeric NC1 segment. A monomeric intermediate with four to six free SH groups and a higher electrophoretic mobility than the final product was observed. Generation of this intermediate from dimers Da and Db follows apparently different routes proceeding either directly or through a dimeric intermediate respectively. The time course of conversion is best described by a mechanism consisting of two (Db) or three (Da) consecutive steps with pseudo-first-order rate constants ranging from 0.14 ms-1 to 0.5 ms-1. Glutathione-catalyzed reoxidation of completely reduced NC1 in the presence of 2 M urea results in a product indistinguishable from native material by ultracentrifugation and electrophoresis pattern. The data suggest that in situ formation of NC1 structures is catalyzed by a small fraction (5-10%) of intrinsic SH groups leading to the formation and stabilization of dimers by rearrangement of disulfide bonds.
authors.affiliation: Biozentrum, Universität Basel, Abteilung Biophysikalische Chemie, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Weber
authors.forename: R
authors.initials: R
authors.lastname: Dölz
authors.forename: R
authors.initials: R
authors.lastname: Timpl
authors.forename: J H
authors.initials: JH
authors.lastname: Fessler
authors.forename: J
authors.initials: J
authors.lastname: Engel
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D003094:Collagen / Q000096:biosynthesis* / Q000302:isolation & purification; D004220:Disulfides / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism; D005260:Female; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D046911:Macromolecular Substances; D008433:Mathematics; D051379:Mice; D008962:Models, Theoretical; D009374:Neoplasms, Experimental / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011487:Protein Conformation
chemical_list: D004220:Disulfides; D046911:Macromolecular Substances; D003094:Collagen; D005033:Ethylmaleimide