Characteristics of prolactin binding to rat liver plasma membranes

Binding sites for prolactin have been identified and characterized in a plasma membrane enriched fraction isolated from livers of mature female rats. By chemical and enzymatic analysis the membrane preparation was shown to have slight contamination with nuclei and endoplasmic reticulum, while mitochondria were not detected. Sidedness analysis indicated that the membrane preparation was largely composed of inside-out vesicles. ¹²⁵I-oPRL prepared by the lactoperoxidase method had a specific activity of 40-60 μCi/μg. Competition studies using iodoprolactin indicated that iodination of the hormone did not affect its affinity for the receptor as compared to the native hormone. Binding of ¹²⁵I-oPRL was inhibited by prolactin from various species including ovine, bovine and rat prolactin while bGH, pACTH and AVP had no effect on binding. The binding of 125 I-oPRL was activated by both bivalent and monovalent cations - bivalent cations exerting a greater effect than monovalent cations. In the presence of 10 mM CaCl₂, binding of ¹²⁵I-oPRL was equal to the binding in the presence of the physiological concentration of NaCI. The association of ¹²⁵I-oPRL with the membrane was a time and temperature dependent process, being maximal at 37°. The dissociation of ¹²⁵I-oPRL was time and temperature dependent only with 150 mM NaCl at 37° while at all other temperatures and in the presence of 10 mM CaCl₂ dissociation was not.observed. The binding of ¹²⁵I-oPRL was strongly influenced by pH with an optimum observed at pH 6.5. Receptor activity was destroyed by pronase and phospholipase C, while neuraminidase increased binding. Treatment of the membranes by RNase and DNase did not effect the binding. Binding of ¹²⁵I-oPRL was inhibited by p-chloromercuribenzoic acid, dithiothreitol, and by brief exposure to high temperatures. Scatchard analysis of the binding of ¹²⁵I-oPRL to receptors indicates that prolactin has a high affinity for its receptorMedicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat