Preparation of biologically active sup 32 P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain

Journal Article · Tue Jun 05 04:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6719107

Osheroff, P L; Ling, V T; Vandlen, R L; Cronin, M J; Lofgren, J A ^[1]

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1. Genentech, Inc., South San Francisco, CA (USA)

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.

• OSTI ID: 6719107
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:16; ISSN 0021-9258; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMP
ANIMALS
ATP
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CENTRAL NERVOUS SYSTEM
CHEMICAL PREPARATION
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DOSE-RESPONSE RELATIONSHIPS
ELECTROPHORESIS
FEMALE GENITALS
GROWTH FACTORS
HORMONES
INSULIN
ION EXCHANGE CHROMATOGRAPHY
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MAN
MASS SPECTROSCOPY
MEMBRANE PROTEINS
MITOGENS
MOLECULAR STRUCTURE
NERVOUS SYSTEM
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HORMONES
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
POLYPEPTIDES
PRIMATES
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SEPARATION PROCESSES
SPECTROSCOPY
SYNTHESIS
UTERUS
VERTEBRATES