Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

from semi-intact cells, sulfate-labeled vesicles were released from pulse-labeled cells, especially after incubation in vitro in the presence of ATP When PC12 cells were pulse labeled 5 min with [ 31 S]sulfate, permeabilized, and incubated 15 min at 37°C, 35 % ofthe total TCA-precipitable radioactivity was released into the S1 from the permeabilized cells if the cells were incubated with an ATP regenerating system (Fig . 2 C, SI) . When Sl was analyzed by velocity sedimentation at higher g force than that used by , most of the membrane-associated TCA-precipitable 31S radioactivity entered the gradient and sedimented in a single peak (fractions 16-22) near the top . The homogeneity is because of uniform sedimentation properties rather than the vesicles reaching equilibrium density, since the distance sedimented was proportional to centrifugation time and force (data not shown) . The radioactivity at the very top of the gradients (fractions 24-26) was probably associated with soluble protein since it could be removed if membranes in the Sl were isolated by centrifuging through a small sucrose pad (Bennett et al ., 1988 ) before application to sucrose velocity gradients (see below) .