Latency of dopamine β-hydroxylase in purified noradrenergic vesicles

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Purified large dense core noradrenergic vesicles isolated from bovine splenic nerve were investigated to determine the percentage of total dopamine β-hydroxylase which existed in a latent or masked form. The effects of the nonionic detergent Triton X-100 in concentrations between 0.0025 and 0.1% were studied. At 0.01%, Triton produced a maximum three-fold increase in accessible dopamine β-hydroxylase activity to reach 0.64μmol octopamine per mg protein per min at 37°C, but released only a third of the enzyme into the soluble phase. About 90% of the noradrenaline, 42% of the phospholipids and 17% of the vesicle protein were released. At 0.05%, Triton produced the same maximum enzyme activity with complete solubilization of the enzyme. All the noradrenaline, 90% of the phospholipids, and 48% of the vesicle protein were released into the soluble phase. Ultrastructural examination of the treated vesicles revealed only partial lysis with 0.01% Triton, while 0.05% produced essentially complete disruption of vesicles into membrane fragments and fine granular matrix contents. Equilibrium centrifugation of the control and treated vesicles on a second sucrose-D 2O density gradient showed that emptied and partially depleted vesicles and released matrix granules redistributed from the heavy vesicle peak to less dense regions of the gradient, including the zone of the light vesicle peak. It is proposed that the latency of dopamine β-hydroxylase activity in this noradrenergic vesicle fraction is best explained by the occurrence of the enzyme as an inaccessible complex in the vesicle matrix which may include phospholipid.