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The World of Protozoa, Rotifera, Nematoda and Oligochaeta

Ref ID : 3650

Simone Eperon and Robert K. Peck; Structural and Biochemical Characterization of Isolated Trichocysts of the ciliate Pseudomicrothorax dubius. J.Protozool. 35(2):280-286, 1988

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Trichocysts of Pseudomicrothorax dubius were ejected by 15% ethanol in phosphate-buffered culture medium (CM) and purified on discontinuous sucrose gradients, in which they concentrated in the lower part of the 27% phase and in the 5% phase. These phases were washed by 15% ethanol in CM, or by CM alone, and pooled. Ejected trichocysts observed by Nomarski interference contrast microscopy and after negative-staining for electron microscopy show a shaft with periodic cross-bands and four opened-out arms, sometimes with electron-dense droplets at both ends of each arm. On SDS-PAGE, trichocysts show ~20 protein bands. The major bands are at 31 and 30 kD (G1), 27 and 26.5 kD (G2), 25 kD, 23 kD, and six bands at 15-20 kD (G3). Minor bands are observed above 30 kD, among them ciliary components which contaminate the trichocyst fraction. The trichocyst banding pattern was reproducible with different ejection media; however, the 30 kD disappeared when the buffered ejection medium contained no added Ca2+ or contained EDTA. When the trichocyst extract is solubilized in sample buffer without 2-mercaptoethanol, the major trichocyst bands are those of G1 and bands at 32.5-35 kD and 41 kD, which appear to be dimers of a few of the G3 proteins. On two-dimensional gels of trichocysts, ~40 acids protein spots are resolved with pI's of 4.6-6.6. On Western blots of two-dimensional gels, glycoproteins were revealed by Concavalin A-peroxidase labeling in three spots of G3, in two spots at 23 kD, in all five spots of G1, and in seven spots over 35 kD.