Use of recombinant DNA derived human relaxin to probe the structure of the native protein.

This report describes the physical, chemical, and biological characterization of recombinant human relaxin (rhRlx) used as a probe to establish the disulfide pairing in native human relaxin. This strategy is necessary since native human relaxin is only available in the nanogram range. The relaxin molecule is composed of two nonidentical peptide chains, an A-chain 24 amino acids in length and a B-chain of 29 amino acids, linked by two disulfide bridges with an additional disulfide linkage in the A-chain. Native relaxin isolated from human corpora lutea was compared to rhRlx by reversed-phase chromatography, partial sequence analysis, mass spectroscopy, and bioassay. The potency of rhRlx was established by its ability to stimulate cAMP from primary human uterine endometrial cells. Native relaxin isolated from human corpora lutea was equipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the complete amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (CysA10-CysA15, CysA11-CysB11, and CysA24-CysB23) were assigned by mass spectrometric analysis of the tryptic peptides and by comparison to chemically synthesized peptides disulfide linked in the two most probable configurations. In addition, the observed amino acid composition and sequence of rhRlx was in agreement with that predicted from the cDNA sequence with the exception that the A-chain amino terminal was pyroglutamic acid. The migration of rhRlx upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was consistent with a monomeric structure, and the identity of the band was demonstrated by immunoblotting.

[1]  A. Chen,et al.  Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay. , 1990, Biochemical and biophysical research communications.

[2]  M. Niwa,et al.  Direct identification of disulfide bond linkages in human insulin-like growth factor I (IGF-I) by chemical synthesis. , 1989, Journal of biochemistry.

[3]  G H Snyder,et al.  Dependence of formation of small disulfide loops in two-cysteine peptides on the number and types of intervening amino acids. , 1989, The Journal of biological chemistry.

[4]  J. Cook,et al.  Structure and activity dependence of recombinant human insulin-like growth factor II on disulfide bond pairing. , 1989, The Journal of biological chemistry.

[5]  Stephen A. Martin,et al.  Collision-induced fragmentation of (M + H)+ ions of peptides. Side chain specific sequence ions , 1988 .

[6]  J. Merryweather,et al.  Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology. , 1988, Biomedical & environmental mass spectrometry.

[7]  K. Titani,et al.  Identification of disulfide-bridged substructures within human von Willebrand factor. , 1987, Biochemistry.

[8]  M. Cronin,et al.  Human relaxin increases cyclic AMP levels in cultured anterior pituitary cells. , 1987, Biochemical and biophysical research communications.

[9]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[10]  K. Biemann,et al.  Characterization by tandem mass spectrometry of structural modifications in proteins. , 1987, Science.

[11]  Andrew J. S. Jones,et al.  Immunoassay for the detection of E. coli proteins in recombinant DNA derived human growth hormone. , 1986, Journal of immunological methods.

[12]  A. Karlin,et al.  Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues. , 1986, The Journal of biological chemistry.

[13]  S. M. McCormack,et al.  The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in rat myometrial cells in culture. , 1985, Endocrinology.

[14]  T. Blundell,et al.  The Conformation of Insulin-Like Growth Factors: Relationships with Insulins , 1985, Journal of Cell Science.

[15]  J. Shine,et al.  Relaxin gene expression in human ovaries and the predicted structure of a human preprorelaxin by analysis of cDNA clones. , 1984, The EMBO journal.

[16]  J. Stewart.  Solid Phase Peptide Synthesis , 1984 .

[17]  S. Shire.  pH-dependent polymerization of a human leukocyte interferon produced by recombinant deoxyribonucleic acid technology. , 1983, Biochemistry.

[18]  J. Shine,et al.  Structure of a genomic clone encoding biologically active human relaxin , 1983, Nature.

[19]  J. Adams.  Heavy metal intensification of DAB-based HRP reaction product. , 1981, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[20]  W. N. Burnette,et al.  "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. , 1981, Analytical biochemistry.

[21]  K. Bhoola,et al.  Modulation of cyclic AMP in isolated rat uterine tissue slices by porcine relaxin. , 1980, The Journal of endocrinology.

[22]  N. Weisbrodt,et al.  The interaction of relaxin with the rat uterus. I. Effect on cyclic nucleotide levels and spontaneous contractile activity. , 1980, Endocrinology.

[23]  S. A. Braddon,et al.  Relaxin-dependent adenosine 6',5'-monophosphate concentration changes in the mouse pubic symphysis. , 1978, Endocrinology.

[24]  G. Abraham,et al.  A technique for the removal of pyroglutamic acid from the amino terminus of proteins using calf liver pyroglutamate amino peptidase. , 1978, Biochemical and biophysical research communications.

[25]  J. Mcdonald,et al.  Relaxin: a disulfide homolog of insulin. , 1977, Science.

[26]  H. Niall,et al.  Primary structure of porcine relaxin: homology with insulin and related growth factors , 1977, Nature.

[27]  I. Holbrook,et al.  A procedure to increase the sensitivity of staining by Coomassie brilliant blue G250-perchloric acid solution. , 1976, Analytical biochemistry.

[28]  M. Bodanszky,et al.  Structure and synthesis of malformin A1 , 1975 .

[29]  R. Hayashi,et al.  Carboxypeptidase from yeast. Large scale preparation and the application to COOH-terminal analysis of peptides and proteins. , 1973, The Journal of biological chemistry.

[30]  H. Matsubara,et al.  High recovery of tryptophan from acid hydrolysates of proteins. , 1969, Biochemical and biophysical research communications.

[31]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.

[32]  F. Sanger,et al.  The disulphide bonds of insulin. , 1955, The Biochemical journal.

[33]  E. F. Graham,et al.  The Effect of Relaxin and Mechanical Dilation on the Bovine Cervix , 1953 .

[34]  S. Moore,et al.  Chromatography of amino acids on sulfonated polystyrene resins. , 1951, The Journal of biological chemistry.

[35]  R. K. Meyer,et al.  THE RELAXATIVE HORMONE OF THE CORPUS LUTEUM. ITS PURIFICATION AND CONCENTRATION1 , 1930 .

[36]  F. Hisaw.  Experimental relaxation of the pubic ligament of the guinea pig. , 1926 .

[37]  E. Canova‐Davis,et al.  Characterization of chemically synthesized human relaxin by high-performance liquid chromatography. , 1990, Journal of chromatography.

[38]  Y. Ovchinnikov,et al.  Complete amino acid sequence of γ‐subunit of the GTP‐binding protein from cattle retina , 1985 .

[39]  E. O'Byrne,et al.  Purification and characterization of porcine relaxin. , 1974, Archives of biochemistry and biophysics.

[40]  C. Hirs.  [6] Determination of cystine as cysteic acid , 1967 .