Effects of dansyl-arginyl-(4'-ethyl)piperidine amide on gonadotropin binding to rat tissues

The effects of dansyl-arginyl-(4'-ethyl)-piperidine amide (DAPA) on the binding of gonadotropins has been investigated. The compound inhibited the binding of hCG, human LH, and porcine LH to rat luteal membranes with an ED50 of 70 microM and the binding of human FSH to rat testicular membranes with an ED50 of 350 microM. Alteration of the substitution at the 4 carbon of the piperidine moiety altered the effect for hCG binding to luteal tissue, the ED50 being lowered with increased chain length (ED50 for H greater than methyl greater than ethyl greater than propyl = isobutyl) and shifted approximately half an order of magnitude for each carbon added. Analysis of equilibrium binding data for hCG to rat luteal membranes showed a decrease in the Ka with increasing DAPA concentrations (2.2 X 10(10), 1.0 X 10(10), and 0.4 X 10(10) M-1 for 0, 50, and 150 microM DAPA, respectively), with little or no effect on the number of sites. At a concentration of 50 microM, the compound did not affect the initial rate of association of the hormone and its receptor, but did change the dissociative behavior. After a short period of association, dissociation was followed in the absence and presence of DAPA. In all cases a biphasic dissociation was observed. The presence of DAPA slowed the rate constant for the fast phase (DAPA-treated, 4.7 X 10(-3) min-1; control, 7.6 X 10(-3) min-1) and reduced the fraction of total hCG undergoing the slow phase of dissociation (DAPA-treated, 60.6%; control, 77.4%). After a long period of association, the dissociation of hCG was monophasic, and the presence of DAPA increased the rate constant from 2.0 X 10(-4) to 4.4 X 10(-4) min-1. The results show that DAPA acted as an inhibitor of gonadotropin binding. The kinetic data suggest a sequential model of hormone binding in which the compound affected a step subsequent to the initial interaction.