DETECTION AND CHARACTERIZATION OF GTP-BINDING PROTEINS ON TONOPLAST OF SPINACIA-OLERACEA

Despite the growing interest in GTP-binding proteins and their role in higher plants, no compartimental analysis has been performed on tonop last, until now. After successive extraction with a cushion of sacchar ose and a glycerol gradient, the tonoplast of Spinacia oleracea was ch ecked by electron microscopy and the purity of the preparations verifi ed by enzyme marker analyses. Guanosine triphosphate (GTP) binding ass ays were carried out on the extract with guanosine 5'[gamma-thio] trip hosphate, [S-35] (GTP gamma(35)S) as the non-hydrolysable substrate. A specific binding was observed and a K-D of 0.3 mM was estimated by di splacement curves. The characteristic enhancement by Mas 7 was also ob served. Two dimensional gel electrophoresis of the GTP-binding test pe rmitted localization of proteins between 20 and 55 kDa which bound spe cifically to GTP gamma(35)S. Immunodetection was performed in the same experimental conditions with an antibody raised against the conserved consensus sequence of the GTP-binding site of the animal G(alpha) sub unit of the heterotrimeric G-Protein. It confirmed the presence of pro teins of 41-44 kDa which correspond to those detected with the GTP-bin ding test. Copyright (C) 1997 Elsevier Science Ireland Ltd.