Purification and properties of chicken growth hormone and the development of a homologous radioimmunoassay

Abstract

Highly purified growth hormone (GH) has been isolated from pituitary glands of chicken (Gallus domesticus), and a specific homologous radioimmunoasssay (RIA) has also been developed. The purified chicken GH was active in the rat tibia bioassay and it gave a dose-dependent response which paralleled that of the bovine GH standard. High pressure liquid chromatography revealed that the purified chicken GH was homogenous. Chicken GH had an R(f) value of 0.2 in disc electrophoresis, and a MW of 26,000 from sodium dodecyl sulfategel elecltrophoresis. The isoelectric point was estimated to be 7.6 by gel isoelectric focusing. The amino acid composition of chicken GH was found to be similar to that of mammalian GH, and the NH 2-terminal amino acid was threonine. Partial sequencing (114 amino acids) of the chicken GH showed 79% homology with bovine GH. An antiserum was developed to the purified chicken GH in a rabbit, and it was used to develop a homologous RIA using 125I-labeled chicken GH as the ligand. The purified chicken GH was iodinated via the lactoperoxidase method to a specific activity of approximately 100 μCi/ug. Plasma from chickens, medium from incubation of pituitary glands, and homogenates of pituitary glands gave parallel dilution-response curves with the chicken GH standard. Mammalian GH, prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) showed no cross-reaction with the 125I-labeled chicken GH. Purified turkey GH showed parallel dose response with the chicken GH, but purified turkey PRL did not cross-react. Chicken FSH and LH also showed no inhibition of binding. The minimum detectable concentration of the assay was 0.93 ng/tube, an the intraassay and interassay coefficients of variation were 9 and 16%, respectively. The specific binding of 125I-labeled chicken GH to a microsomal fraction isolated from chicken liver was identified, and the specific binding was generally low (1-4%). Turkey PRL, and chicken LH and FSH showed no inhibition of the 125I-labeled chicken GH hepatic binding and the ontogeny of the hepatic GH receptor binding site in male and female chickens was examined.