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Structural and Biochemical Characterization of Isolated Trichocysts of the Ciliate Pseudomicrothorax dubius 1
Author(s) -
EPERON SIMONE,
PECK ROBERT K.
Publication year - 1988
Publication title -
the journal of protozoology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 0022-3921
DOI - 10.1111/j.1550-7408.1988.tb04344.x
Subject(s) - ciliate , chemistry , organelle , differential interference contrast microscopy , centrifugation , electron microscope , chromatography , biophysics , biology , biochemistry , microscopy , paleontology , physics , optics
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 57% 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 (G 1 ), 27 and 26.5 kD (G 2 ), 25 kD, 23 kD, and six bands at 15–20 kD (G 3 ). 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 Ca 2+ or contained EDTA. When the trichocyst extract is solubilized in sample buffer without 2‐mercaptoethanol, the major trichocyst bands are those of G 1 and bands at 32.5–35 kD and 41 kD, which appear to be dimers of a few of the G 3 proteins. On two‐dimensional gels of trichocysts, ˜40 acidic 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 G 3 , in two spots at 23 kD, in all five spots of G 1 , and in seven spots over 35 kD.