Influx of Ca2+ into isolated secretory vesicles from adrenal medulla Influence of external K+ and Na+

Secretory vesic1es from adrenal medulla contain catecholamines, nuc1eotides and proteins, all of which are released into the extracellular fluid during exocytosis. Adrenal medullary secretory vesic1es also contain high concentrations of Ca'+ [1]. The mechanism of the aecumulation of Ca 2+ into the vesicles is largely unknown and the experimental data eoncerning the uptake of Ca'+ into isolated secretory vesicles are contradictory. It has been reported that secretory vesicle membranes are impermeable to Ca'+ [2], that secretory vesicles take up Ca 2+ independently of ATP [3] and that they possess an ATP-stimulated uptake system [4,5]. In earlier work relatively impure and unstable seeretory vesicle fractions were used for the determination of Ca 2+ -uptake. We have developed a method to isolate highly purified and stable secretory vesicles from bovine medulla [6]. With these vesic1es we repeated earlier Ca'+ -uptake experiments and found that: (i) The vesic1es take up <sCa2+ in K+-containingmedia; (ü) 4SCa2+ uptake is abolished in the presence ofNa+; (üi) nie Ca 2+ content of isolated secretory vesic1es is increased when incubated with Ca 2+ in media containing K+, but not in media containing Na +

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