Ca2+ binding to Chromaffin Vesicle Matrix Proteins

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.