Isolation of the membranes from secretory organelles (trichocysts) of Paramecium tetraurelia

Abstract

We present for the first time a method for isolation of the membranes of extrusive organelles (trichocysts) from sterile culture of different strains of Paramecium tetraurelia. First, trichocysts are isolated according to a new method (Glas-Albrecht, R. and Plattner, H. (1990) Eur. J. Cell Biol. 53, 164–172) with high purity and yield. Then the organelles are subjected to osmotic swelling. Since trichocysts then easily ‘decondense’ and entangle membranes, these cannot be isolated directly by centrifugation, but only by passage through a filter and subsequent centrifugation. Purity of membrane fractions is analysed by electron microscopy and SDS-PAGE, combined with silver staining or, after biotinylation, by avidin-peroxidase labelling. Molecular masses resolved in our gels are in a range from ≤ 15 to ≥ 105 kDa. Main bands obtained with nd9–28°C trichocyst membranes (most bands also being common to wild type trichocysts) are of about 16.5, 19–21, 27–29, 33–34, 44–45 (strong), 47–48 (strong), 57, 61, 65 (strong), 68–71, 75, 81, 94–95 (strong), 104 and ≥ 110kDa, from a total of approx. 23 bands resolved. There is no remarkable occurrence of dominant protein bands from trichocyst contents (‘trichynins’), though these might represent up to 103-times more of the total trichocyst proteins. The ratio of phospholipid/protein is approx. 0.2 mg/mg. The methodology developed might also be valuable for the isolation of extrusome membranes from some other protozoan species.