Reiffen, F. U.; Gratzl, Manfred
Ca2+ binding to Chromaffin Vesicle Matrix Proteins. Effect of pH, Mg2+, and Ionic Strength.
In: Biochemistry, Vol. 25: pp. 4402-4406
Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl,
M. (1985) Biochemistry 24, 7760-77651. In order to explore the nature of these bonds, we analyzed the
binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles.
The dissociation constant at pH 7 is 50 pM (number of binding sites, n = 180 nmol/mg of protein) for
Ca2+-protein bonds and 15 pM (n = 0.8 pmol/pmoi) for Ca2+-ATP bonds. When the pH is decreased
to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity
of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70
pM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present
within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the
affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+
we found a dissociation constant of 340 pM and after addition of 35 mM K+ a dissociation constant of 170
pM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized
by a Kd of 240 pM and after addition of 15 mM Na' by a Kd of 340 pM. The similar affinity of Ca2+
for protein and ATP, especially at pH 6, in media of increased ionic strength and after addition of Mg2+,
points to the possibility that the intravesicular medium determines whether Ca2+ is preferentially bound
to ATP or the chromaffin vesicle matrix proteins. Purified chromogranin A, after sodium dodecyl sulfate-
polyacrylamide gel electrophoresis, stains with a carbocyanine dye ("Stains-all") and, following blotting
onto nitrocellulose, binds to 45Ca2+. A spectrophotometric analysis of dye binding to chromaffin vesicle
matrix proteins revealed a strong absorption band at 615 nm for the dye-protein complex. Since the observed
spectral changes were unaffected by the presence of Ca2+ (100 pM free), the sites interacting with the dye
and Ca2+ must be regarded as different.