In vivo biosynthesis of two subunit forms of dopamine beta-hydroxylase in rat brain

In vivo biosynthesis of the 2 subunit forms of dopamine beta-hydroxylase (DBH) was examined in the rat brain. 35S-methionine was injected into the noradrenergic neurons of the locus coeruleus (LC) using a stereotactic device. Several hours later, newly synthesized 35S-Met-labeled DBH was immunoprecipitated and quantitated. Both Mr = 77,000 (77K) and 73,000 (73K) subunit forms were present in near-equal proportions after 4 hr of labeling, and these were indistinguishable from those isolated from rat PC12 pheochromocytoma cells by electrophoretic mobility. Both forms sedimented with the vesicular subcellular fraction of LC homogenates, and a portion of the 73K form could be released by hypotonic lysis of these vesicles. The 77K form predominated in the first 30 min labeling period, while the 73K form appeared more slowly over the next several hours. By 16 hr, the 73K form comprised about 2/3 of the total 35S-Met-labeled DBH present. Inhibition of protein synthesis with puromycin 30 min after 35S-Met injection into the LC did not prevent the subsequent appearance of the 73K form, suggesting that this subunit form was the product of posttranslational modification of the 77K subunit form in a fashion similar to that seen in PC12 cells. Also, newly synthesized 35S-Met-labeled DBH that underwent axonal transport from the LC to the anterior hypothalamus was predominantly the 73K subunit form. A single injection of the catecholamine-depleting drug reserpine (10 mg/kg, i.p.) produced a 17-fold increase in the relative synthesis of DBH 2 d later without affecting the proportion of its 2 subunit forms.