Isolation by calcium-dependent translation to neutrophil-specific granules
of a 42-kD cytosolic protein, identified as being a fragment of annexin XI
C Sjolin and C Dahlgren
Phagocyte Research Laboratory, Department of Medical Microbiology and
Immunology, University of Goteborg, Sweden.
Secretion of neutrophil granules is dependent on calcium, but at the same
time this process is regulated differently for each type of granules. 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 micromol/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 partly 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.
Volume 87,
Issue 11,
pp. 4817-4823,
06/01/1996
Copyright © 1996 by The American Society of Hematology
