Isolation by calcium-dependent translocation to neutrophil-specific granules of a 42-kD cytosolic protein, identified as being a fragment of annexin XI

Abstract

Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granule. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti-cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 μmol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pertly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N- terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.