Characterization of [125I]-iodo-ovine prolactin and evaluation of prolactin receptor assay methods.
In an attempt to establish more rigorous conditions for assay of prolactin receptor, a study of the preparation, recovery and specific binding of iodoprolactin was conducted. Averaging the results of 20 preparations starting with 5 micrograms prolactin preparation and iodinated by a lactoperoxidase method, an average of 0.62 +/- 0.12 micrograms of the iodinated prolactin was recovered in a form capable of binding to excess rat liver membrane preparations, corresponding to between 24 and 51% of the total recovered iodinated prolactin. Based on preliminary amino acid analysis and N-terminus determination, 53% of a weighed amount of ovine prolactin was determined to be protein with the N-terminal amino acid being consistent with homogeneity. The percent specific binding assay (single point assay) and Scatchard analysis were tested for their ability to give assay results in a direct relationship to sample receptor. Scatchard analyses were determined to be a superior method to the percent specific binding assay. Using commonly employed conditions for the percent specific binding assay, the relative amounts of receptor found in increasing amounts of sample were significantly less than that found by Scatchard analysis in all likelihood due to the use of subsaturating concentrations of iodohormone. As more hormone was added per assay tube, the relationship of sample receptor to the results of the single point assay improved, but the results never equaled those of Scatchard analyses. Thus, many workers who have used the percent specific binding assay under nonsaturating conditions have underestimated the relative receptor contents of samples.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D008099:Liver; D011388:Prolactin / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000032:analysis*; D011981:Receptors, Prolactin; D012756:Sheep
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011981:Receptors, Prolactin; D011388:Prolactin