Micromethod for the determination of free and total prolactin receptors: measurement of receptor levels in normal and malignant mammary and prostate tissues.

A sensitive micromethod for the determination of free and total prolactin receptors in normal or malignant tissues has been developed. Positive and negative quality controls are incorporated in the procedure. Either whole tissue or the pellet fraction remaining from tissue that had undergone processing for estrogen receptors can be used. Crude microsomal and plasma membrane fractions obtained by homogenization and differential centrifugation are incubated with labeled prolactin in the presence or absence of increasing amounts of unlabeled hormone. The labeled ligand is prepared by a stoichiometric iodination procedure in which one atom of iodine-125 is incorporated into one molecule of the hormone, resulting in an intact labeled prolactin with a high specific activity of 170-186 muCi/micrograms (1 Ci = 37 GBq). Human prolactin labeled by this procedure has much greater specific binding capacity to various rat tissues than does iodinated rat prolactin. This technique permits an accurate measurement of prolactin receptors in as little as 50 micrograms of membrane protein. Highest levels of free and total prolactin receptors were found in the liver of 60-day-old female rats that served as a positive control. Liver of immature 21-day-old male rats, devoid of prolactin receptors, was used as a negative control. The amount of detectable free receptors was dependent on the level of circulating plasma prolactin. In 3-day postpartum lactating rats with high prolactin levels in plasma, all prolactin receptors in the mammary glands were found to be occupied, and no free receptors could be detected. When these receptors were desaturated from the endogenous prolactin by exposure to 3 M MgCl2, one class of receptors in a high quantity (1.75 nmol/mg of protein) and with a moderate affinity (Kd = 6.41 X 10(-9) M) was detected. A similar type of receptor was found in the mammary glands of rats at midpregnancy and of cycling adult female rats. In malignant rat mammary tissue, however, fewer receptors (27 pmol/mg of protein) but with a very high affinity (Kd = 6.8 X 10(-14) M) were detected. Normal ventral and dorsolateral rat prostate contained two classes of prolactin receptors (Kd = 3.46 X 10(-10) M and 1.93 X 10(-8) M). In the cancerous rat prostate, however, only one of these two classes of receptors was detected, and the number was smaller.