Abstract

The distribution of dopamine β-hydroxylase (EC 1.14.21) and catecholamines in the adrenal glands of rabbits has been determined by differential and isopycnic centrifugation at various times after treatment with insulin. Fractionation of the subcellular components by differential centrifugation demonstrated that all the dopamine β-hydroxylase bound to the storage vesicle membrane was retained 3 hr after insulin-induced secretion and that only the soluble dopamine β-hydroxylase within the storage vesicle matrix was released. Twenty four hours after insulin treatment there were changes in the distribution of the membrane-bound enzyme which suggest that partial restoration of storage vesicles had already occurred and that at least some of the membranes of the evacuated vesicles were destroyed.

Isopycnic centrifugation of the crude storage vesicle fraction obtained from adrenal glands 3 hr after insulin administration showed marked decreases in total dopamine β-hydroxylase and catecholamine content but no changes in their relative amounts in those fractions of the sucrose density gradient containing intact storage vesicles and no changes in the buoyant densities of the storage vesicles. These studies indicate that those vesicles which participated in the secretory response released their entire soluble content in an "all-or-none" fashion.

Twenty-four hours after insulin treatment both the dopamine β-hydroxylase and catecholamine content of the glands was still markedly depleted. The buoyant density of the storage vesicles was less than that of vesicles obtained from adrenal glands of untreated rabbits, but the dopamine β-hydroxylase to catecholamine ratio of the vesicles was the same as that of controls. Forty-eight hours after insulin treatment the dopamine β-hydroxylase levels had recovered to normal but the catecholamine content was only 35% of control values. The buoyant density of the storage vesicles suggested that there was a larger proportion of vesicles with lighter than normal densities, and the dopamine β-hydroxylase to catecholamine ratio was 3 times normal values. Glands examined 96-144 hr after insulin treatment were indistinguishable from glands of untreated animals. These studies indicate that during recovery the formation of storage vesicles is not the rate-limiting step, and that there is a considerable lag between the formation of the storage vesicles and recovery of the catecholamine stores.