Partial purification and characterization of bovine mammary gland prolactin receptor.
Prolactin (PRL) receptors from the mammary gland of the lactating cow were solubilized with 3-[(3-cholamidopropyl)dimethylamonio]-1-propane sulfonate (CHAPS). Affinite chromatography on human growth hormone (hGH) coupled to Affi-Gel 10 resulted in over 500-fold purification, as compared to microsomal fractions. Scatchard analysis of the binding of hGH indicated an increase in the affinity constant of 2.5-fold after solubilization and of further 2-fold after the affinity purification. The specific binding activity of the affinity-purified fraction was 9000 fmol hGH/mg protein. Complexes of Triton X-100-solubilized receptors with [125I]hGH were analyzed by gel filtration on Sephadex G-150, in the presence of Triton X-100. A minor fraction of the complexes eluted as high molecular weight (Mr) aggregates, whereas a major fraction eluted as a 150 kDa peak. Assuming a contribution of approximately 30% to the Mr by the bound detergent and a hormone: receptor ratio of 1:1 in the complex, a Mr of 80-85 kDa can be calculated for the receptor molecule. Affinity labelling of the receptor with [125I]hGH revealed a Mr of 37 +/- 0.5 kDa (n = 7) for the binding subunit. Specific high Mr aggregates were also observed after crosslinking; however, the size of the labelled species was not affected by reducing agents. Homologous and heterologous competitive binding studies with ovine PRL (oPRL) or hGH revealed a considerably higher affinity for hGH as compared to oPRL. The competitive displacement patterns obtained with oPRL or hGH as tracers were similar, indicating that both hormones bound to the same receptor sites with different affinities. A similar difference in affinity was retained by the affinity-purified receptors.
D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D013006:Growth Hormone / Q000378:metabolism; D007457:Iodine Radioisotopes; D007774:Lactation / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000032:analysis*; D008970:Molecular Weight; D011247:Pregnancy; D011388:Prolactin / Q000378:metabolism; D011981:Receptors, Prolactin / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D007457:Iodine Radioisotopes; D011981:Receptors, Prolactin; D011388:Prolactin; D013006:Growth Hormone