Chemical cross-linking of porcine luteinizing hormone: location of the cross-link and consequences for stability and biological activity.
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.
van Dijk
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Department of Biochemistry and Molecular Biology, M. D. Anderson Cancer Center, Houston, Texas 77030.
Ward
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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
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