Partial characterization of mitochondrial G proteins in adrenal cells

摘要

Four low molecular mass G proteins have been identified in mitochondrial membranes from bovine adrenal cortex. These proteins (referred to as proteins 1 to 4) showed molecular masses of 28, 27, 26 and 24 kDa with isoelectric points (pI) of 8.1, 5.6, and 6.3 respectively for proteins 1, 2 and 4. Protein 3 was shown to be heterogeneous, with isoelectric points of 5.0-6.1. Proteins were identified by binding of [α-~(32)P]guanosine triphosphate (GTP) after separation by 12% SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose. Competitive binding by unlabelled competing nucleoside phosphate ligands showed specificity for guanosine triphosphate (GTP) and guanosine diphosphate (GDP) with little binding of guanosine monophosphate and no detectable binding with adenosine nucleoside phosphates. Binding was less than 10% with 100-fold excess GDP and GTP which showed equal intensities of binding. Inhibition of binding by 1000-fold cytidine triphosphate and uridine triphosphate was approx. 10%. Magnesium (Mg~(2+)) stimulated binding of GTP by all four proteins. The effect of Mg~(2+) was essentially the same for proteins 1, 2 and 3, while protein 4 was less sensitive to Mg~(2+) at concentrations < 10~(-3) M. Centrifugation of sonicated mitochondrial membranes through sucrose density gradients showed the presence of all four proteins in contact points. The presence of lower concentrations (expressed per mg protein) of the proteins in inner and outer membranes suggests that either small amounts of these membranes are part of contact points as presently prepared or that the proteins occur in contact points and to a much smaller extent in inner and outer membranes. It is proposed to examine a possible role for these proteins in transport of cholesterol from outer to inner mitochondrial membranes.