Ultrastructural and cytochemical characterization of adrenal medullary plasma membrane vesicles and their interaction with chromaffin granules.

Plasma membrane vesicles obtained by density gradient centrifugation of bovine adrenal medullary homogenates were analyzed by electron microscopic methods, including negative staining, ultrathin sections and freeze-fracture replicas. Rapid freezing showed the intramembrane structure of plasma membrane vesicles to be distinct from that of other organelle membranes, such as chromaffin granules. Cytochemical demonstration of acetylcholinesterase (EC 3.1.1.7) activity on most membrane profiles confirmed that plasma membrane vesicles are derived predominantly from plasma membranes. About half of the plasma membrane vesicles were smaller than 0.15 micron and almost none larger than 0.55 micron. Practically all were composed of single shells. Most vesicles were impermeable to cytochemical markers of the size of Ruthenium red (Mr 800) and none were permeable to markers larger than 40 kDa. Surface charge probes, concanavalin A binding and endogenous actin decoration with heavy meromyosin indicated that the major fraction of plasma membrane vesicles is oriented right-side-out. A minor population with opposite orientation could also be detected. Isotonic ionic media caused vesicle aggregation in suspensions of plasma membrane vesicles and chromaffin granules. Freeze-fracturing always revealed clusters of membrane-intercalated particles at the sites of contact between aggregated membranes.