ISSN:
1550-7408
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
Notes:
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 (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 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 G3, in two spots at 23 kD, in all five spots of G1, and in seven spots over 35 kD.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1550-7408.1988.tb04344.x
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