Characteristics of histidyl-proline diketopiperazine [cyclo (His-Pro)] binding sites in the rat liver were studied using (3)H-labeled cyclo (His-Pro). Scatchard analysis suggested that the rat liver membrane had a single binding site with an apparent dissociation constant (K(d)) of 92×10(-9)M. Pretreatment of membrane preparations with soybean trypsin inhibitor increased cyclo(His-Pro) binding, and the binding activity was sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for cyclo (His-Pro) binding. Thiol reagents reduced binding activity, suggesting that the thiol group might be an important constituent of the cyclo (His-Pro) binding site. Cross-reactivities of TRH, TRH analogues, L-His and L-Pro were very low (0.2-9%). Cyclo (His-Pro) binding of female rat liver was markedly lower than that of male rat liver. Scatchard analysis showed that the sex difference in cyclo (His-Pro) binding was due to different binding capacity. Cyclo (His-Pro) binding of castrated male rat liver was significantly decreased. Testosterone replacement raised the binding to the control level. These findings indicate that testosterone is an important factor in the regulation of cyclo (His-Pro) binding in the rat liver. Intraperitoneal injections of cyclo(His-Pro) (15 mg/kg/day) to mice for 3 days caused a significant loss of cyclo (His-Pro) binding sites in the liver. This loss depended on the decrease in the number of binding sites and not on the change in affinity. The cyclo (His-Pro)-mediated loss was reversible, and the specific binding returned to the original level within 3 days of the cessation of cyclo (His-Pro). These results suggest that cyclo (His-Pro) participates in regulating its own binding sites in the liver. They also indicate that specific binding sites for cyclo (His-Pro) in the rat liver have similar properties to the receptors for other polypeptides.
histidyl-proline diketopiperazine [cyclo (His-Pro)]