AJP - Cell Physiology, Vol 262, Issue 4 C907-C915, Copyright © 1992 by American Physiological Society
Binding and aggregation of pro-atrial natriuretic factor by calcium
G. Thibault and A. F. Doubell
Laboratory of Cell Biology of Hypertension, University of Montreal, Quebec, Canada.
Analysis of atrial secretory granule content by sodium dodecyl sulfate-gel
electrophoresis followed by a 45Ca2+ overlay assay indicates that a 17,000
protein binds 45Ca2+. This protein, which can be immunostained by atrial
natriuretic factor (ANF) antiserum, corresponds to proANF. Ca2+ binding is
proportional to the amount of proANF and pH dependent. Generation of
ANF-(1-98) by thrombin digestion of proANF does not affect Ca2+ binding.
Blocking the carboxyl groups of proANF and the use of NH2-terminal
fragments bearing those carboxyl groups demonstrated that the
Ca(2+)-interaction site is probably located within the highly acidic
portion (11-30) of the propeptide. Ca2+ binding to proANF induces its
aggregation that can be verified by sedimentation. ProANF aggregation is
Ca2+ dependent, being optimal at 10 mM, partially pH dependent, and greatly
increased by high concentrations of proANF. However, because of its
relatively low-binding affinity, Ca2+ can be substituted by other divalent
cations such as Sr2+, Ba2+, or Mg2+. The high level of Ca2+ in atrial
secretory granules and the aggregation of proANF in the presence of Ca2+
suggest a possible involvement of these physicochemical properties in the
condensed state of the matrix of secretory granules. Indeed, detergent
solubilization of the membrane of the secretory granules in presence of
Ca2+ resulted only in a partial dissolution of the dense core matrix. We
therefore postulate that, in the Golgi complex, proANF and Ca2+ associate
to form a condensed aggregate that helps package secretory material into
secretory vesicles.
