title: Characterization of a biotin-conjugated ovine prolactin ligand.
abstract: Ovine prolactin was biotinylated with N-hydroxysuccinimidobiotin. Biotinylation was proportional to the molar ratio of reactants. Gel electrophoresis of the biotinylated derivative revealed little or no change in migration, but isoelectric focusing showed an acidic shift when compared to oPRL. Biotinylated ovine prolactin (B-oPRL) was detected by anti-oPRL antiserum and avidin-fluorescein-isothiocyanate (FITC) on protein blots. Competitive binding assays using 125I-B-oPRL and 125I-oPRL revealed: (a) similar dissociation constants and ID50 values for binding to anti-oPRL antibodies; (b) similar dissociation constants and maximum binding values for binding to liver membrane preparations; and (c) similar dissociation curves for displacement by several pituitary hormones. In contrast, binding of biotinylated oPRL to Nb2 node cells was reduced by approximately 70% and its bioreactivity was only 10% of that of oPRL. Our results indicate that biotinylation of oPRL does not alter its binding characteristics as measured by radioimmunoassay and radioreceptor assay using hepatic lactogenic receptors, but decreases its binding and bioreactivity when measured in Nb2 lymphoma cells. Assuming that N-hydroxysuccinimidobiotin interacts with reactive free amino groups of oPRL, our results suggest that these groups are essential for binding and bioreactivity of the molecule in the Nb2 lymphoma cell system.
authors: Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis.
authors: E
authors: E
authors: Michel
authors: J A
authors: JA
authors: Parsons
mesh_terms: D000818:Animals; D001360:Avidin; D001710:Biotin / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D007525:Isoelectric Focusing; D008024:Ligands; D011388:Prolactin / Q000032:analysis*; D011863:Radioimmunoassay; D011869:Radioligand Assay; D012756:Sheep
chemical_list: D007457:Iodine Radioisotopes; D008024:Ligands; D001360:Avidin; D001710:Biotin; D011388:Prolactin