Detection of low molecular mass GTP-binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells

Abstract

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [α- 32P]GTP in the presence of Mg 2+. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [α- 32P]GTP was specific and dependent on Mg 2+. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.