Photoaffinity labelling of steroid-hormone-binding glutathione S-transferases with [3H]methyltrienolone. Inhibition of steroid-binding activity by the anticarcinogen indole-3-carbinol

The identification and characterization of steroid-hormone-binding glutathione S-transferases (GST) were undertaken using photoaffinity-labelling techniques. Irradiation of mouse liver cytosol, in the presence of 50 nM-[3H]methyltrienolone, resulted in the specific affinity labelling of five proteins. One of these proteins, designated MBP27, had an approximate molecular mass of 27 kDa under denaturing conditions and was induced by treatment of mice with either 2(3)-t-butyl-4-hydroxyanisole (BHA) or phenobarbital (PB). An additional affinity-labelled protein, MBP25, which was not detected in untreated mouse cytosol, was induced in the liver cytosols from BHA- and PB-treated mice. The molecular masses of these proteins and their induction by BHA and PB suggested that they may be steroid-hormone-binding GST subunits. Irradiation of mouse liver cytosol in the presence of [3H]methyltrienolone, followed by immunoprecipitation using GST-specific antibodies established that both GST mu and GST alpha bind [3H]methyltrienolone and both contribute to the affinity-labelled protein designated MBP27. GST Ya1 Ya1, an alpha class GST that is not expressed in untreated mouse liver but is induced by BHA and PB, was also found to bind [3H]methyltrienolone and is identical with the affinity-labelled protein designated MBP25. Experiments were undertaken next to assess the effects of the anticarcinogenic plant compound indole-3-carbinol (I3C) on GST-mediated steroid hormone-binding using the photoaffinity labelling techniques. Treatment of mice with I3C resulted in the induction of immunoreactive GST mu and GST Ya1 Ya1. However, the steroid-binding activity of these proteins in vitro was severely inhibited by the acid-condensation products of I3C that are generated in the stomach after ingestion. These results suggest that I3C may inhibit GST-mediated steroid-binding activity which could contribute to the anticarcinogenic activity of this compound.

Participation of concanavalin A binding sites in the interaction between Trypanosoma cruzi and macrophages

Untreated mouse peritoneal macrophages as well as macrophages treated with concanavalin A (ConA) were incubated in the presence of untreated or ConA-treated epimastigotes and trypomastigotes of Trypanosoma cruzi. Treatment of epimastigotes or trypomastigotes with ConA increased or decreased their uptake by macrophages, respectively. Treatment of their macrophages with ConA reduced by 70% and increased by five times the ingestion of epimastigotes and trypomastigotes, respectively. These results are discussed in relation to previous studies on the mobility of ConA receptors in the membrane of the parasite. Using fluorescein- or ferritin-labelled ConA we observed that ConA binding sites located on the plasma membrane of macrophages are internalized during endocytosis of T. cruzi, and observed in association with the membrane of the endocytic vacuole. Vacuoles without parasites showed a uniform distribution of ConA binding sites, while these sites were distributed in patches in vacuoles containing parasites. These results, in association with others previously reported, suggest the involvement of glycoproteins and/or glycolipids localized on the cell surface of T. cruzi and macrophages during the T. cruzi-macrophage interaction.