Sedimentation characteristics of noradrenergic vesicles from rat interscapular brown adipose tissue

Abstract

Rat interscapular brown adipose tissue (IBAT) was homogenized to release the noradrenergic vesicles present in its dense sympathetic innervation. The vesicles were then studied by several sedimentation techniques using noradrenaline (NA) and dopamine beta-hydroxylase (DBH) as markers. Sixty-three percent of the DBH activity and 29% of the NA in homogenates (0.25 M sucrose, 5 mM Tris, pH 7.4 at 21 degrees C) of IBAT from 28 degrees C acclimated rats sedimented in the microsomal fraction (226 600 X gmax, 60 min). Differential sedimentation of the microparticulate DBH in a low-speed supernatant fraction of the homogenate indicated at least two distinct populations of microparticles with average sedimentation coefficients of 80 +/- 11 and 255 +/- 42 S (4 degrees C) and containing, respectively, about 65 and 35% of the sedimentable DBH. Upon isopycnic, sucrose density centrifugation of the resuspended microsomal fraction, DBH peaked at a density of 1.091 but extended as a broad shoulder up to a density of about 1.19. During rate zonal centrifugation of the resuspended microsomal fraction on sucrose density gradients, microparticulate DBH and NA separated into slow and fast moving components. The modal density of the slow moving component upon isopycnic recentrifugation was 1.092, while the fast moving one, similarly treated, became almost equally distributed over a range of densities from 1.12 to 1.19. For the slow moving component, NA and DBH relative to protein were, respectively, 6.5 and 23 times more concentrated than in the IBAT homogenate. On the basis of its measured sedimentation characteristics, the slow moving component would correspond to vesicles having a calculated diameter of 66 nm. The data thus indicate that in IBAT, DBH and NA can be separated into two distinct populations of sedimentable particles. Whether or not these correspond to the small and large dense-cored vesicles observed by ultramicroscopy of IBAT remains to be demonstrated.