Binding and cross-linking of iodinated rat prolactin to rat hepatic prolactin receptor.
Basal parameters for binding and cross-linking of 125I-rat prolactin (rPRL) to lactogenic (PRL) binding species present in crude membrane fraction (CMF) or detergent-solubilized preparations of rat liver have been investigated. (1) The highest specific binding to CMF was obtained with an incubation time of 50 h at 20 degrees C and with a 50 mM potassium phosphate buffer adjusted to pH 8.5. (2) Cross-linking of 125I-rPRL to binding sites in CMF with disuccinimidyl suberate (DSS) showed the autoradiographic appearance of an Mr 40,000 binding species. (3) No specific binding or cross-linking of rPRL was seen in Triton X-100-solubilized CMF. This is probably due to Triton X-100-induced changes in the physical properties of rPRL. (4) Specific binding of 125I-rPRL was detected in CHAPS-solubilized CMF. Following cross-linking the autoradiographic appearance of a binding species with an Mr value of 40,000 was shown. 125I-hGH was cross-linked to three PRL binding species with Mr 82,000, 40,000 and 35,000 in CHAPS-solubilized preparations. (5) In Golgi-enriched low-density membrane preparation 125I-rPRL was cross-linked to Mr 82,000, 40,000 and 35,000 species. It is proposed that the inability of rPRL to be cross-linked to Mr 82,000 and 35,000 species present in CHAPS-solubilized preparation is the result of CHAPS-induced changes of rPRL binding properties and low solubilizing capacity of CHAPS. (6) In conclusion, this study shows that also the iodinated endogenous hormone, rat prolactin, and not only hGH identifies high and low molecular forms of the rat liver prolactin receptor.
Haldosén
L A
LA
Department of Medical Nutrition F60, NOVUM, Huddinge University Hospital, Sweden.
Gustafsson
J A
JA
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D003432:Cross-Linking Reagents; D005260:Female; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011981:Receptors, Prolactin / Q000378:metabolism*; D012995:Solubility; D013388:Succinimides
D002793:Cholic Acids; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011981:Receptors, Prolactin; D013388:Succinimides; D011092:Polyethylene Glycols; D011388:Prolactin; D017830:Octoxynol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C019358:disuccinimidyl suberate