Bioactivity and immunoreactivity of rat pituitary prolactin submitted to various extraction procedures

Pituitary glands of rats, injected with estrogen to increase the prolactin (PRL) storage in secretory granules, were submitted to various extraction procedures for prolactin. Homogenization and centrifugation of pituitary tissue, in Tris-HCl buffer, pH 7.3, yielded a small amount of radioimmunoassayable prolactin, which increased remarkably after extraction in alkaline pH, disruption of granular membranes with Lubrol and specially after treatment with 2.5 mol/l urea. Nb2 lymphoma cell and pigeon crop sac bioassay (BA) revealed higher levels of bioactivity after extraction in Tris buffer, pH 7.3, in comparison to RIA, with BA/RIA ratios of 1.4 and 2.3, respectively. Bioassays of pituitary PRL extracted with Lubrol and alkaline medium, were less effective in quantifying PRL than RIA. Treatment of pituitary homogenates with urea produced the highest levels of PRL by both BA and RIA, with BA/RIA ratios close to 1. The thorough depolymerization of big PRL effected by urea produced the release of monomeric subunits which allows a complete quantification of the total content in the pituitary gland.