Characteristics of the binding of 32P-labelled human relaxins to the human fetal membranes.
The two human relaxin genes termed H1 and H2 are expressed in the choriodecidua and placenta and have been proposed to act via specific receptors as local modulators of collagenolysis in the fetal membranes. Such receptors have been inferred, but not demonstrated, from studies of the effect of adding exogenous relaxin to these tissues. Thus conditions were optimized for the binding of 32P-labelled human relaxin H2 to membrane-enriched particulate fractions of human fetal membranes, amnion and chorion, with adhering decidua. The membrane protein concentration was optimal at 250 micrograms, when incubated at 27 degrees C for 60 min, at pH 7.5 with Mn2+ and Mg2+ ion concentrations of 2.0 mM. Incubation of membrane particulate fractions with increasing amounts of labelled relaxin H2 suggested the presence of a single class of binding sites with an affinity constant (Ka) of 2.15 nM. The binding was primarily to the chorion and decidua with very little to the amnion layer. The competition for binding of the 32P-labelled human relaxin H2 with unlabelled relaxin H2 gave an IC50 of 28 pM, while an IC50 of 60 pM and 280 pM was obtained for relaxin H1 and porcine relaxin respectively. In contrast, unlabelled guinea-pig relaxin inhibited this binding by only 10% even at a 1000-fold greater concentration than H2, and human recombinant insulin failed to inhibit even at a million-fold concentration of unlabelled relaxin H2. Relaxins H2 and H1 can readily displace the binding of either 32P-labelled human relaxins H1 or H2 and gave very similar displacement curves.(ABSTRACT TRUNCATED AT 250 WORDS)
Garibay-Tupas
J L
JL
Pacific Biomedical Research Center, University of Hawaii at Manoa, Honolulu 96822, USA.
Maaskant
R A
RA
Greenwood
F C
FC
Bryant-Greenwood
G D
GD
D005321:Extraembryonic Membranes / Q000378:metabolism*; D005260:Female; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012065:Relaxin / Q000378:metabolism*
C095067:RLN1 protein, human; C095068:RLN2 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C033395:relaxin receptors; D012065:Relaxin