Overlay blot identification of GTP-binding proteins in mitochondria from human placenta

In this study, an overlay blot method was used to identify GTP-binding proteins in fractions of human placenta. Human placenta were fractionated by centrifugation into preparations containing (1) mitochondria, (2) nucleoli and (3) microsomes, plasma membrane and cytosol. GTP-binding proteins were detected by overlay blot using alpha32P-GTP. Proteins of 23 and 25 kDa were identified in all fractions and GTP binding was higher in the presence of 1.0, 2.5, 5.0 and 10.0 mM MgCl2 as compared to equivalent concentrations of CaCl2. In mitochondrial preparations binding of alpha32P-GTP to 23 and 25 kDa was displaced significantly by GDP and GTP but not ADP or ATP. Fractions containing microsomes, plasma membrane and cytosol displayed two labelled bands of 14 and 18 kDa that were not present in other fractions. These data indicate that the placenta contains specific GTP-binding proteins of molecular weights that are consistent with the small monomeric GTP binding protein family (18-36 kDa). Two of these are located in the mitochondria and may regulate the function of these organelles in the placenta.