title: Chemical cross-linking of porcine luteinizing hormone: location of the cross-link and consequences for stability and biological activity.
abstract: To study the interaction between the subunits of LH and determine which amino acid residues are involved in this interaction, porcine and ovine LH (pLH and oLH) were cross-linked with 0.02 M 1-ethyl-(3-(3-dimethylaminopropyl)carbodiimide to generate one specific intersubunit cross-link. The cross-linked hormone was separated from the noncross-linked dissociated subunits by gel filtration on a Sephadex G-75 column. The position of the cross-link in cross-linked pLH (X-pLH) was determined by sequencing peptide fragments that were generated by digestion with endoproteinase Arg-C. In accordance with previous data for bovine LH, the position of the cross-link was between alpha-Lys49 and beta-Asp111, indicating that these residues are at the subunit-subunit interface. The biological activity of cross-linked hormone was tested by radioreceptor binding assay. The receptor-binding activity of X-pLH was slightly reduced to 84%, suggesting that the conformational stability of X-pLH is similar to that of pLH as a result of the introduction of the covalent cross-link. The receptor-binding activity of X-oLH was decreased by approximately 30%, which we attribute to the formation of multiple cross-links within the ovine molecule, making the molecule more rigid, and to oligomeric forms, resulting from multiple intermolecular cross-links, that are not able to bind to the testicular LH receptor. This observation implies that the oLH has more amino-carboxyl functional groups that are sterically susceptible to carbodiimide cross-linking than does pLH under the reaction conditions used.
authors.affiliation: Department of Biochemistry and Molecular Biology, M. D. Anderson Cancer Center, Houston, Texas 77030.
authors.forename: S
authors.initials: S
authors.lastname: van Dijk
authors.forename: D N
authors.initials: DN
authors.lastname: Ward
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives*; D007986:Luteinizing Hormone / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011974:Receptors, LH / Q000378:metabolism; D013552:Swine; D013737:Testis / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011974:Receptors, LH; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D007986:Luteinizing Hormone; D005022:Ethyldimethylaminopropyl Carbodiimide