Affinity-purification of concanavalin A-binding ciliary glycoconjugates of starved and feeding Tetrahymena thermophila.
Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.
Driscoll
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Department of Biochemistry, Microbiology and Molecular Genetics, University of Rhode Island, Kingston 02881, USA.
Hufnagel
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D000818:Animals; D002846:Chromatography, Affinity; D002923:Cilia / Q000737:chemistry* / Q000378:metabolism; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006001:Glycoconjugates / Q000302:isolation & purification* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D012098:Reproduction; D015398:Signal Transduction; D012995:Solubility; D016808:Tetrahymena thermophila / Q000737:chemistry* / Q000502:physiology
D006001:Glycoconjugates; D008565:Membrane Proteins; D010455:Peptides; D003208:Concanavalin A