A calcium-stimulated alkaline phosphatase associated with synaptic vesicles

Synaptic vesicles isolated from bovine cerebral cortex were found to contain alkaline phosphatase activity towards p-nitrophenylphosphate and a-naphthyl phosphate, but not towards pyridoxal phosphate. The enzyme had an apparent molecular weight of 125,000 and co-purified with the synaptic vesicles in parallel with the specific neurotransmitter content and with the loss of contaminating components, whereas the major phosphatase which was present in the brain homogenate, with an apparent molecular weight of 140,000 purified away. The optimal pH for the enzyme activity on p-nitrophenylphosphate was 9.8. At this pH the activity was 33.4 nmol/mg protein/min, and the apparent K_m value was 0.31 ± 0.05mM. The pH dependency of the synaptic vesicle phosphatase activity towards p-nitrophenylphosphate differed from that of the Ca²⁺/Mg²⁺-dependent ATP hydrolysis by the synaptic vesicles. Upon mild digestion of lyzed vesicles with trypsin, phosphatase activity was reduced whereas the ATPase activity was retained suggesting that the phosphatase and the ATPase are two different enzymes. The phosphatase was reversibly inhibited by ethyleneglycol bis (aminoethyl ether) N,N′-tetraacetic acid (EGTA) and activity was restored by the addition of an equimolar amount of Ca²⁺. Magnesium ions could restore only 30% of the activity. The activity of the synaptic vesicle phosphatase was not affected by o-phenanthroline, zinc ion or by cAMP. Tetranitromethane inactivated the enzyme irreversibly, whereas phenylmethanesulfonylfluoride diisopropylfluorophosphate and p-hydroxy-mercurybenzoate inhibited the activity partially. The enzyme did not have a diesterase activity. Adenosine mono-, di- and triphosphate inhibited the p-nitrophenylphosphatase activity and were also hydrolyzed by the vesicle preparation. However, the different kinetic parameters obtained with the nucleotide as inhibitors or as substrates suggest that additional enzymes are involved in the hydrolysis of the adenine nucleotides in vesicle preparation.