Localization of different steps in noradrenaline synthesis to different fractions of a bovine splenic nerve homogenate

Abstract

Experiments were carried out to determine to what extent the synthesis of nor-epinephrine in bovine splenic nerve tissue can proceed in the highspeed sedimentable particulate fraction that contains the specific norepinephrine storage vesicles. Various fractions of a homogenate of this tissue were incubated in a potassium phosphate medium together with radioactively labeled precursors of norepinephrine. The first two steps in the synthesis, the ring hydroxylation of tyrosine and the decarboxylation of dopa, were shown not to require the pres -ence of this particulate fraction. On the other hand, this fraction was an absolute prerequisite for the last step, the [beta] -hydroxylation of dopamine. No significant formation of catechol compounds from tyrosine could be demonstrated on incubation with this particulate fraction after it had been washed and resuspended in potassium phosphate. Some dopa decarboxylase activity remained in the particulate fraction even after washing. The results support the view that in this tissue only one enzyme involved in noradrenaline synthesis, dopamine [beta]-hydroxylase, is truly located in the above-mentioned particles, possibly inside the membrane of the noradrenaline-storing vesicles. Tyrosine hydroxylase and dopa decarboxylase appear to be located outside these particles. Dopa decarboxylase shows some association with the particulate fraction studied, and might be, to some extent, bound to be outer aspect of the same particles which contain the [beta] -hydroxylase, or could just be accumulated in the perivesicu-lar parts of the probably highly organized axoplasm.