Identification of the histidine residues of hemopexin that coordinate with heme-iron and of a receptor-binding region.
Rabbit hemopexin cDNA was cloned from a rabbit liver lambda gt11 cDNA expression library using a mixture of five monoclonal antibodies raised against rabbit hemopexin, and the entire rabbit hemopexin sequence was determined. The heme-binding domain I of rabbit hemopexin (Smith, A., and Morgan, W. T. (1984) J. Biol. Chem. 259, 12049-12053) contains only 4 histidine residues which are conserved in rabbit, human, rat, and mouse hemopexin. The 2 axial heme-iron coordinating histidine residues, identified by Edman microsequencing and amino acid analyses of chemically modified domain I and isolated fragments of domain I, are the conserved histidine residues at positions 56 and 127 of the mature rabbit protein. The epitope recognized by JEN-14 (a monoclonal antibody which specifically reacts with domain I and blocks the hemopexin-receptor interaction (Morgan, W. T., Muster, P., Tatum, F. M., McConnell, J., Conway, T. P., Hensley, P., and Smith, A. (1988) J. Biol. Chem. 263, 8220-8225) was shown to lie between residues 122 and 142 by Western blotting of protease-digested domain I and transposon-insertion mutants of domain I expressed in a plasmid vector system. The location of this epitope near the heme-binding histidine residue 127 is compatible with a transport mechanism in which the release of heme from hemopexin is accompanied by a concomitant transfer of heme to the hemopexin receptor or the membrane heme-binding protein (Smith, A., and Morgan, W. T. (1985) J. Biol. Chem. 260, 8325-8329).
Morgan
W T
WT
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110.
Muster
P
P
Tatum
F
F
Kao
S M
SM
Alam
J
J
Smith
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA; D000939:Epitopes; D006418:Heme / Q000378:metabolism*; D006466:Hemopexin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006639:Histidine / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018000:Receptors, Peptide; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C047453:hemopexin receptor; D006418:Heme; D006639:Histidine; D004247:DNA; D006466:Hemopexin; D007501:Iron