Origin of the Bicarbonate Stimulation of Torpedo Electric Organ Synaptic Vesicle ATPase

The origin of the stimulation by bicarbonate of the ATPase found in cholinergic synaptic vesicles isolated from Torpedo californica electric organ was studied. Given in descending order of potency, the anions formate, malonate, acetate, thiosulfate, cyanate, biphosphite, biselenite, and bisulfite also stimulate to lesser extents. The anion channel blockers 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulfonic acid (SITS) and 4,4′‐diisothiocyanostil‐bene‐2,2′‐disulfonic acid (DIDS) inactivated the ATPase at micromolar concentrations, but there was no indication that the reagents acted to block an anion channel. The LineweaverBurk plot of the basal ATPase activity was biphasic, indicating high‐ and low–affinity states with Kms of 0.0098 ′ 0.0013 and 1.2 ′ 0.1 mM for Vmaxs of 52 ′ 2 and 82 ′ 9 nmol/min/mg protein, respectively. It became nearly linear in the presence of bicarbonate, indicating conversion of all of the ATPase to one high affinity‐high velocity state with a Km of 0.054 ′ 0.009 mM and a Vmax of 232 ′ 0.051 nmol/min/mg protein. The pH activity profile for basal ATPase was bell‐shaped and optimal between pH 6 and 8. The bicarbonate stimulation was maximal at about pH 7. The ATPase could be solubilized in 1.6% Lubrol WX with full retention of the bicarbonate stimulation. Although ouabain and oligomycin act at high concentrations to inhibit bicarbonate stimulation of the ATPase, there is no contamination by the Na+‐K+ ATPase, mitochondrial ATPase, or other common ATPases. The study suggests that bicarbonate acts to stimulate the ATPase by acting directly on it at an allosteric site on the outside of the synaptic vesicle.