Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain.
Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.
Osheroff
P L
PL
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080.
Ling
V T
VT
Vandlen
R L
RL
Cronin
M J
MJ
Lofgren
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D001921:Brain / Q000378:metabolism*; D002584:Cervix Uteri / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007163:Immunosorbent Techniques; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012065:Relaxin / Q000378:metabolism* / Q000494:pharmacology; D014599:Uterus / Q000187:drug effects / Q000378:metabolism*
D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D012065:Relaxin; D000242:Cyclic AMP; D011494:Protein Kinases