Spontaneous Vesiculation of Chromaffin Granules in vitro
نویسندگان
چکیده
It is now fairly well established that the catecholamines and ATP stored in the adrenalmedullary chromaffin granules are released by exocytosis, a process involving fusion of the chrome-granule membrane to the external limiting membrane of the chromaffin cell and extrusion of the core. The proteins and the lipids of the granule membrane are not released, however, and it has been suggested on the basis of electron-micrographic evidence that the granule membrane breaks up into smaller vesicles after the granule contents have been released (Smith & Winkler, 1972). We report here on changes in the structure of granules in vitro under conditions in which the catecholamine stores of the granule are depleted and we comment on these changes as they apply to current models of exocytosis. Bovine adrenal chromaffin granules were prepared essentially as described by Phillips (1973). All granule preparations used for the light-scattering experiments were first filtered through Millipore or Sartorius membrane filters (450nm pore size) to remove mitochondria and granule aggregates (Formby et af., 1972). Granules filtered through 300nm-pore filters produced qualitatively the same changes in light-scattering. Mitochondria] contamination, as judged by the activity of monoamine oxidase present, was very low. Filtration through a 45Onm-pore filter greatly decreased the intensity of light scattered by a mitochondria sample taken from the preparative density gradient, and the mitochondria which did pass through the filter showed very little change in light-scattering under the conditions used for these experiments. When 2 0 0 ~ 1 of the granule suspension (protein concentration l-2mg/ml) in 0.30.4~-sucrose at 0°C was resuspended in 20ml of 0.3 M-sucros~lOrn-Hepes$ buffer, pH7.2, at 18-20°C, the intensity of the light scattered at the angles 30°, 45", and 135" (I,,,, Z45 and I,,,) decreased with time and finally stabilized after several hours (Fig. la). For any given granule preparation, the time-course of the change was independent of the presence of 2 m ~ N a + or K+ ions in the sucrose, although it did proceed faster when 0.16~-KC1 was used as a solvent. When the granules are rapidly diluted 50-100 times into 0.3 M-sucrose they lose 80 % of their ATP and catecholamine stores within 1 min; the remaining 20% is stable over the course of 1 h (Fig. lc). Thus the loss of catecholamine and ATP is too rapid to account for the bulk of the change in light-scattering by itself, although it may be represented in the fast primary falling phase. When granules suspended in 0.3~-sucrose are rapidly diluted into 10mM-Hepes buffer, pH7.2, the granules lyse and release 99.8 % of the lowmolecular-weight components and most of the soluble protein (Fig. lc). The membrane 'ghosts' appear electron-micrographically as hollow vesicles of a wide size range. When the granules are resuspended in lorn-Hepes buffer, pH7.2, the scattering intensity and dissymmetry after the first 2-3min does not change with time. The complete angular light-scattering spectrum of the granules resuspended in 0.3 M-sucrose is approximately the same as that for the lysed granules in buffered water when both samples are re-examined after 18-24 h, which suggests that the granules resuspended in 0.3M-sucrose undergo structural changes similar to those produced by lysis, only more slowly. The biochemical and morphological evidence suggests that the changes observed in light-scattering are due to changes in granule structure after the granules have lost their stores of small molecules. The angular spectrum of scattered light changed too rapidly to attempt to determine the change in size and shape of the particles directly by the Zimm method (Zimm, 1948 ; Geiduschek & Holtzer, 1958). It was possible, however, to measure
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