Solid-phase synthesis of ovine Leydig cell insulin-like peptide--a putative ovine relaxin?

J Pept Res. 1999 May;53(5):542-7. doi: 10.1034/j.1399-3011.1999.00060.x.


The primary structure of ovine Leydig cell insulin-like peptide (Ley I-L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B-chain, an Arg-X-X-X-Arg sequence exists within the Ley I-L B-chain although it is located four residues towards the C-terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I-L suggested that the peptide might possess a relaxin-like function. Ovine Ley I-L was assembled by Fmoc-solid-phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain-combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin-like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I-L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I-L and relaxin, we prepared a synthetic analogue of ovine Ley I-L containing a single replacement of B-chain residue 12, His, with Arg. This was found to possess significant relaxin-like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I-L is similar to that of relaxin and highlighting the key requirement for the five-residue sequence, Arg-X-X-X-Arg, to be present in position B12-16 for characteristic relaxin activity.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Cardiotonic Agents / pharmacology
  • Heart Atria / drug effects
  • Heart Rate / drug effects
  • Hydrogen-Ion Concentration
  • Insulin
  • Isometric Contraction / drug effects
  • Isoproterenol / pharmacology
  • Male
  • Mass Spectrometry
  • Molecular Sequence Data
  • Myocardial Contraction / drug effects
  • Proteins / chemical synthesis*
  • Proteins / pharmacology
  • Proteins / physiology
  • Rats
  • Rats, Sprague-Dawley
  • Relaxin / pharmacology
  • Relaxin / physiology*
  • Sheep
  • Solutions
  • Stimulation, Chemical


  • Cardiotonic Agents
  • Insulin
  • Leydig insulin-like protein
  • Proteins
  • Solutions
  • Relaxin
  • Isoproterenol