Effects of Protein-Modifying Reagents on Brain Tryptamine Binding Sites: Possible Involvement of a Thiol Group in Temperature-Induced High-Affinity [³H]Tryptamine Binding Sites

Abstract

To investigate the biochemical nature of temperature-induced highaffinity [³H]tryptamine binding sites, we subjected whole rat brain synaptic membranes to treatment with various protein-modifying reagents and examined the subsequent [³H]tryptamine binding properties of the membranes. Pretreatment of the membrane preparations with NEM, NBS, PCMB, PAPMA and MA, but not with iodoacetamide, DTT, glutathione and cysteine, reduced the [³H]tryptamine binding. In addition, to at least approx. 10^-4 M, the inactivation properties of NEM, PCMB, PAPMA and MA, except for NBS, were temperature-dependent. Furthermore, it was revealed that the Scatchard plot of [³H]tryptamine binding in membranes pretreated with these thiol reagents conformed to a curved line, as well as in the case of the control membranes. Nonlinear regression analysis of these data showed that NEM decreased the B_max values of both the high and low affinity binding sites with no significant alteration in the K_D values, whereas PCMB, PAPMA and MA increased only the K_D value of the high affinity sites, accompanying the decrease of the B_max values of both sites. These results indicate that the temperature-induced high-affinity [³H]tryptamine binding molecule(s) is a thiol protein.