Purification of monkey prolactin from culture medium: biochemical and immunological characterization.
Serum-free culture medium, previously incubated with dispersed monkey pituitary cells, provided a relatively uncontaminated source for extraction and purification of 15 mg of monkey prolactin. N-terminal group analysis of this preparation, M21GB, produced predominantly leucine. The amino acid composition closely resembles that of both human and sheep prolactin. M21GB monkey prolactin migrates in 10% polyacrylamide with sodium dodecyl sulfate (SDS) as a single band with a molecular weight of about 23,000. Multiple bands typical of prolactins are seen with nondenaturing polyacrylamide disc electrophoresis. M21GB contains less than 1% growth hormone, incorporates radioactive iodine with a specific activity of 15 microCi/micrograms and specifically binds to the anti-human prolactin serum-3 provided by the National Hormone and Pituitary Program (B spec/total = 23%). M21GB does not compete in a linear fashion with iodinated human prolactin-16 for the human prolactin antiserum, but M21GB does compete in a linear fashion with iodinated M21GB with the same antiserum. Monkey serum, pituitary homogenate, and culture medium containing unknown levels of monkey prolactin are not parallel with NIAMDD-HPrl-RP1 in the human prolactin assay, but are parallel when M21GB is used as the reference preparation and for iodination. Finally, antisera to M21GB were generated in rabbits which are specific for monkey and human prolactin and which can be used for radioimmunoassay or immunocytochemistry. In summary, serum-free medium from primary cultures of dispersed monkey pituitaries provided a quantitity of monkey prolactin which promoted biochemical analysis and production of a specific antiserum. This culture system may be a unique and ongoing source for extraction of significant quantities of monkey prolactin suitable for investigative use.
D000596:Amino Acids; D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008253:Macaca mulatta; D010902:Pituitary Gland / Q000378:metabolism; D011388:Prolactin / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits / Q000276:immunology; D051381:Rats; D012756:Sheep; D013552:Swine
D000596:Amino Acids; D003470:Culture Media; D007106:Immune Sera; D011388:Prolactin