Chapter 21 Studies of Isolated Secretion Granules of the Rabbit Parotid Gland: Ion-Induced Changes in Membrane Integrity

Abstract

Publisher Summary This chapter discusses the possible involvement of phospholipase activities in membrane–membrane interactions. The tissue used for the experiment reported in the chapter was the parotid salivary gland of the rabbit. The parotid salivary gland of the rabbit comprises a homogeneous population of secretory cells, each containing numerous apically located secretion granules (diameter 1.0–1.2 μm) that collectively occupy ∼35% of the cellular volume. In the experiment, secretion granules were isolated and then the purified fractions were obtained after discontinuous sucrose gradient centrifugation in 2 M sucrose buffered with 40 mM potassium phosphate containing 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.2. The sucrose concentration was subsequently reduced to 0.4 M by the continuous addition (for ≥1 hour at 4°C using a peristaltic pump) of 0.25 M buffered sucrose. The diluted suspension was subjected to low-speed centrifugation at 2,000gav for 25 minutes, and the pelleted granules were gently resuspended for further studies in fresh 0.4 M buffered sucrose. The intactness of the granule suspension at this point was determined by measuring the amount (expressed as percentage of total) of α-amylase, a secretory protein normally packaged in the granule content, which is sedimentable during centrifugation of ≤60 μL suspension for 1.5 minutes at 8,000gav (Eppendorf 3,200 centrifuge). The latter conditions ensure complete granule sedimentation, and suspensions were found to be 82–92% intact at this stage.