title: Further characterization of structural determinants of rabbit hemopexin function.
abstract: To further identify structural features of the hemopexin molecule important for its heme transport function, a fragment of the heme-binding domain (residues 1-213, Mr 35 kD, domain I) of rabbit hemopexin was obtained after digestion with subtilisin. Both apo- and heme-domain I were cleaved by subtilisin, and the subtilisin-digested form of domain I (called SD-DI) was shown by microsequencing to have been cleaved at Asp 22 forming a 30 kD subfragment lacking the conserved histidine residue at position 7 and the N-linked oligosaccharide at Asn 9. The 5 kD peptide cleaved from domain I is not disulfide linked to domain I and can be removed by membrane ultrafiltration. SD-DI retains the ability of domain I to bind heme, to associate with the other functional domain of hemopexin (domain II), and to interact with the hemopexin receptor on mouse Hepa cells. Moreover, although the heme complex of SD-DI is less thermostable than native heme-domain I, like heme-domain I, heme-SD-DI is stabilized to a large extent when associated with domain II. These results show that the conserved His 7 residue is not involved in heme binding by hemopexin and that residues 1-22 of hemopexin and the N-linked oligosaccharide at Asn 9 are not essential for either receptor binding or interdomain interactions. Nevertheless, these N-terminal residues of hemopexin do contribute significantly to the overall stability of the hemopexin molecule and the interdomain interactions necessary for receptor recognition.
authors.affiliation: Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110.
authors.forename: P
authors.initials: P
authors.lastname: Muster
authors.forename: F
authors.initials: F
authors.lastname: Tatum
authors.forename: A
authors.initials: A
authors.lastname: Smith
authors.forename: W T
authors.initials: WT
authors.lastname: Morgan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006418:Heme / Q000378:metabolism; D006466:Hemopexin / Q000737:chemistry*; D008969:Molecular Sequence Data; D011817:Rabbits; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000494:pharmacology
chemical_list: D006418:Heme; D006466:Hemopexin; D013381:Subtilisins