Oxytocin binding sites in lactating rabbit mammary gland

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Abstract
Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Kd and binding capacity values were as follows: crude, Kd = 1.83 .times. 10-9 M, capacity = 670 fmol/mg protein; purified, Kd = 2.8 .times. 10-9 M, capacity = 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. Preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor-hormone complex. This complex remained in the supernatant even after centrifugation at 210,000 .times. g for 30 min. This solubilized complex was shown to be oxytocin specific.