Binding and aggregation of pro-atrial natriuretic factor by calcium.
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.
Thibault
G
G
Laboratory of Cell Biology of Hypertension, University of Montreal, Quebec, Canada.
Doubell
A F
AF
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006325:Heart Atria; D006801:Humans; D008854:Microscopy, Electron; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure; D009994:Osmolar Concentration; D011498:Protein Precursors / Q000378:metabolism*
D011498:Protein Precursors; D009320:Atrial Natriuretic Factor; D002118:Calcium