Studies with carbodiimide-cross-linked derivatives of bovine lutropin. II. Location of the cross-link and implication for interaction with the receptors in testes.

We have studied subunit-subunit interactions in bovine lutropin (bLH) through characterization of carbodiimide-cross-linked derivatives (EDCbLH). Spectrophotometric titrations indicated that an additional tyrosine residue ionizes abnormally in EDCbLH compared to native bLH. Furthermore, only five out of seven tyrosines in the cross-linked derivative were ionized at pH 13. Treatment with tetranitromethane resulted in the nitration of four tyrosines in native bLH but only about three tyrosines in EDCbLH. The near and far W circular dichroic spectra of cross-linked derivatives at pH 7.0 are quite similar to those obtained for native bLH. At pH 3.0, the near W CD spectrum of bLH is very nearly equal to the algebraic sum of the spectra of its isolated subunits. Under identical conditions, EDCbLH partially retains a broad negative band in the region 270 to 290 nm indicative of shielded tyrosine residues. Tryptic peptide maps of performic acid-oxidized native and cross-linked bLH were prepared by two-dimensional paper mapping. Four peptides, present in maps of bLH, were absent in maps of the cross-linked derivative. Three of these were peptides derived from residues 47 through 55 of the LY subunit while a single peptide was from the COOH terminus of the fi subunit. A unique peptide was isolated from maps of EDCbLH. Compositional analysis of these peptides allowed the conclusion that the major amide cross-link in EDCbLH has been formed by covalent coupling of lysine 49 (a highly conserved residue from the (Y subunit) and aspartic acid 111 (a residue from the hormone-specific fi subunit conserved only in molecules with LH-like activity). Using banding patterns on sodium dodecyl sulfateurea polyacrylamide gels as criteria for cross-linking, it was possible to demonstrate that subunits of several other glycoprotein hormones including human lutropin (hLH), human follitropin (hFSH), human chorionic gonadotropin (hCG), human thyrotropin (hTSH), and bovine thyrotropin (bTSH) could be covalently linked by exposure to the water-soluble carbodiimide. Examination of the COOH-terminal regions of the fl subunits of the glycoprotein hormones (beyond residue 100 in the bLH sequence) has shown that they contain residues which are highly conserved for a given activ-