Properties of Gonadotropin Receptors in the Cell Membranes of Bovine Corpus Luteurn”

The isolated cell membranes from bovine corpora lutea bound lz51-human chorionic gonadotropin (HCG) with high affinity and specificity. The specific binding was saturable with respect to the amounts of membrane protein and 12jIHCG added. Approximately 8.4 x 10-l” M 124-HCG saturated all the receptor sites in 1.0 mg of membrane protein. The specific binding of 12sI-HCG was strong and not readily reversible in nature. The binding of 129-HCG was inhibited in the presence of unlabeled HCG or luteinizing hormone (LH) in a dose-dependent manner. Prostaglandin El, which mimicked LH in the stimulation of bovine corpus luteum adenylate cyclase and steroidogenesis, had no affinity for HCG-LH binding sites. The rate constants for association and dissociation, 5.9 X lo6 M-‘s-l and 1 .l x 10h3 s-l were measured at 38”. The dissociation constant, 1.9 x lo-lo M, was compared with the value of 1.2 X 1OWo M which was obtained from equilibrium data at 38”. Similar values for the dissociation constants (1.9 X lo-lo M at 38” uersus 2.4 X lo-lo M at 22’) were obtained from the rate data at 38” and 22”. The free energy change for the dissociation of lZ51-HCG from the receptor complex was calculated to be +14.1 Cal per mole. Maximum specific binding of 12”I-HCG occurred at pH 6.2 from 30-38” in contrast to the maximum for nonspecific binding at pH 4.0. Brief exposure of the membranes (15 min) to temperatures of 50-90” resulted in irreversible losses of 1251-HCG binding. Various divalent and monovalent cations in concentrations of 10-l M or higher markedly decreased binding. Estradiol-17P and progesterone either in combination or alone (lop9 M to lop5 M) had no effect on 1251-HCG binding. Among several nucleotides tested, only 1 to 2 mM ATP, AMP, adenosine cyclic 3’: 5’.monophosphate, CTP, and guanosine cyclic 3’: 5’.monophosphate (cGMP) have significantly inhibited lz51-HCG binding; CTP and cGMP being most effective. These nucleotides also increased the dissociation of bound hormone when added at various times during incubation. Incubation of membranes with proteolytic enzymes resulted in a marked loss of lZ51-HCG binding. Neuraminidases had either no effect or increased the binding by 20%. Phospholipase A and C markedly decreased 1251-HCG binding.