Abstract
The bulk of Prolactin (PRL) in rat pituitary gland stored as big molecular forms in secretory granules is mostly excluded from radioimmunoassay (RIA) determinations because secretory granules remain intact after tissue homogenization. Also big PRL is little immunoreactive and must be deaggregated to monomers to allow a complete detection by RIA. Different dissociating agents, used to render PRL monomers soluble, were tested at various temperatures and pH conditions. Incubations of pituitary homogenates in buffers at neutral pH yield consistent levels of PRL, but the sole alkalinization of the media increases significantly the radioimmunoassayable PRL content. No significant increase was detected with EDTA and thiols. The 2.5 M urea was the most effective extraction agent increasing about 7-fold the quantification of PRL by RIA. Extraction of PRL with urea was enhanced at pH 9.0 and at 4 C and this combination constitutes the method of choice for a complete extraction of pituitary PRL.
Similar content being viewed by others
References
Torres A.I., Haggi, E.S., Aoki A. Isolation of two different pools of pituitary prolactin. Acta Endocrinol. (Copenh.) 107: 25, 1984.
Torres A.I, Aoki A. Subcellular compartmentation of prolactin in rat lactotrophs. J. Endocrinol. 105: 219, 1985.
Haggi E., Aoki A. Prolactin content in rat pituitary gland, RIA of prolactin after different extraction procedures. Acta Endocrinol. (Copenh.) 97: 338, 1981.
Zanini A., Giannattasio G. Polyacrylamide gel electrophoresis of rat anterior pituitary gland after different extraction procedures. J. Endocrinol. 53: 177, 1972.
Mena F., Martinez-Escalera G., Clapp C., Aguayo D., Forray C., Grosvenor C.E. A solubility shift occurs during depletion-transformation of prolactin within the lactating rat pituitary. Endocrinology 111: 1086, 1982.
Lawson D.M., Haisenleder D.J., Gala R.R., Moy J.A. The occurrence of pituitary prolactin depletion-transformation in lactating rats: dependence on strain of rats, homogenization conditions and method of assay. J. Endocrinol. 113: 71, 1987.
Niswender G.D., Chen C.L., Midgley A.R. Jr, Meites J., Ellis S. Radioimmunoassay for rat prolactin. Proc. Soc. Exp. Biol. Med. 130: 793, 1969.
Lorenson M.Y., Robson D.L., Jacobs L.S. Detectability of pituitary PRL and GH by immunoassay is increased by thiols and suppressed by divalent cations. Endocrinology 112: 1880, 1983.
Lorenson M.Y., Miska S.P., Jacobs L.S. Molecular mechanism of prolactin release from pituitary secretory granules. In: Mena F. & Valverde-R. C. M. (Eds.), Prolactin secretion: a multidisciplinary approach. Academic Press, New York, 1984, p. 141.
Jacobs L.S., Lorenson M.Y. Extractability of rat pituitary prolactin: rat and bovine prolactins are stored differently. In: Mac Leod R.M., Thorner M.O., Scapagnini U. (Eds.), Prolactin basic and clinical correlates. Liviana Press, Padova, 1985, p. 455.
Giannattasio G., Zanini A., Meldolesi J. Molecular organization of rat prolactin granules. J. Cell Biol. 64: 246, 1975.
Carty S.E., Johnson R.G., Scarpa A. Electrochemical proton gradient in dense granules isolated from anterior pituitary. J. Biol. Chem. 257: 7269, 1982.
Boeri E., Rippa M. The effect of urea on flavocytochrome b2. Arch. Biochem. Biophys. 94: 336, 1961.
Staples S.J., Reithel F.J. Evidence for an active-inactive subunit complex in jack bean urease. Arch. Biochem. Biophys. 174: 651, 1976.
Simpson R.B., Kauzmann W. The kinetics of protein denaturation. I. The behavior of the optical rotation of ovalbumin in urea solutions. J. Am. Chem. Soc. 75: 5139, 1953.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Torres, A.I., Prada, M.I. & Aoki, A. Extraction of rat pituitary prolactin for radioimmunoassay. J Endocrinol Invest 13, 205–208 (1990). https://doi.org/10.1007/BF03349541
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03349541