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

Journal Article · Tue Jun 18 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00238a026· OSTI ID:5025823

Canova-Davis, E; Kessler, T J; Lee, P J; Fie, D T.W.; Griffin, P; Stults, J T; Rinderknecht, E ^[1]; Wade, J D ^[2]

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1. Genetech, Inc., San Francisco, CA (United States)
2. Univ. of Melbourne, Parkville, Victoria (Australia)

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 euqipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the completer amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (Cys{sup A10}-Cys{sup A15}, Cys{sup A11}-Cys{sup B11}, and Cys{sup A24}-Cys{sup B23}) 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.

• OSTI ID: 5025823
• Journal Information: Biochemistry; (United States), Vol. 30:24; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PEPTIDE HORMONES
AMINO ACID SEQUENCE
RECOMBINANT DNA
MOLECULAR STRUCTURE
ELECTROPHORESIS
INSULIN
LIQUID COLUMN CHROMATOGRAPHY
MAN
ANIMALS
CHROMATOGRAPHY
DNA
HORMONES
MAMMALS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PRIMATES
PROTEINS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques