TY - JOUR
T1 - Solid-phase synthesis of ovine Leydig cell insulin-like peptide - A putative ovine relaxin?
AU - Dawson, Nicola F.
AU - Tan, Y. Y.
AU - Macris, M.
AU - Otvos, L.
AU - Summers, Roger J.
AU - Tregear, Geoffrey W.
AU - Wade, John D.
PY - 1999
Y1 - 1999
N2 - 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.
AB - 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.
KW - Chronotropic and inotropic activity
KW - Ovine Leydig cell insulin-like protein
KW - Piperazine
KW - Relaxin
KW - Solid-phase peptide synthesis
UR - http://www.scopus.com/inward/record.url?scp=0032997059&partnerID=8YFLogxK
U2 - 10.1034/j.1399-3011.1999.00060.x
DO - 10.1034/j.1399-3011.1999.00060.x
M3 - Article
C2 - 10424349
AN - SCOPUS:0032997059
VL - 53
SP - 542
EP - 547
JO - Journal of Peptide Research
JF - Journal of Peptide Research
SN - 1397-002X
IS - 5
ER -