Structure of chicken hypothalamic luteinizing hormone-releasing hormone. I. Structural determination on partially purified material.

Abstract

Studies on partially purified chicken hypothalamic luteinizing hormone-releasing hormone (LH-RH) utilizing chromatography, region-specific antisera, enzymic inactivation, and chemical modification have established that the peptide is structurally different from mammalian hypothalamic LH-RH.Gel filtration chromatography demonstrated that chicken LH-RH is of the same molecular size as the mammalian decapeptide.Chicken LH-RH eluted earlier than mammalian LH-RH on reverse phase high performance liquid chromatography.Cation exchange chromatography (CM32 carboxymethylcellulose and high performance liquid chromatography) and isoelectric focusing established that chicken LH-RH is less positively charged than the mammalian peptide.Binding studies of chicken LH-RH utilizing five different region-specific antisera raised against mammalian LH-RH demonstrated that the structural difference resides in amino acid residue 8 (arginine).This finding was confirmed by measurement, with appropriate antisera, of changes in immunoreactivity after cleavage with proteolytic enzymes and chemical modification of specific amino acid residues.The lower isoelectric point of chicken LH-RH (7.3) relative to that of the mammalian peptide (9.1) is compatible with the substitution of a neutral amino acid for arginine at position 8: Structural studies on mammalian LH-RH have shown that the side chain of Arg-8 is in close vicinity to the side chains of His-2 and Tyr-5 and that these side chains are linked by hydrogen bonds and form a combined unit important for biological action (Shinitzky, M., and Fridkin, M. (1976) Biochim.Biophys.Acta 434, 137-143).A likely neutral amino acid substitution compatible with these structural requirements is glutamine.Moreover, of the neutral amino acids, the probability of glutamine replacing arginine (or vice versa) is high (Dayhoff, M. O., Eck, R. V., and Park, C. M. (1972) in Atlas of Protein Sequence and Structure (Dayhoff, M. O., ed) pp.89-99, National Biomedical Research Foundation, Washington, D. C.).We therefore synthesized [Gln 8 ]LH-RH and found it to have chromatographic, immunological and biological properties identical with the natural chicken peptide.