Abstract:
The cell cortex (or pellicle) of "Tetrahymena" is composed of three closely associated
layers: the cell membrane, the alveolar sacs, and the epiplasm. The epiplasm is the
innermost layer of the cortex and is thought to be analogous to the membrane skeleton
found in eukaryotic cells, since it consists largely of filamentous proteins responsible for
maintaining cell stability and shape. Furthermore, the epiplasrnic layer provides a means
by which the cell can regulate the location and distribution of structures along its surface.
Low salt extraction of Triton-potassium iodide (TKI) cortical residues is an efficient
means by which three membrane skeletal proteins (band A (235 kDa), band C (125 kDa),
and a 23 kDa protein) can be isolated. Previous immunofluorescence studies have localized
bands A and C in the cell cortex and irnrnunoblots have demonstrated that these proteins are
major components of the membrane skeleton in "Tetrahymena".
In this study, polyclonal antibodies were raised in rabbits against three membrane
skeletal proteins isolated by low salt extraction of TKI cortical residues: band A and C, and
a 23 kDa protein. Immunoblots were performed to characterize the number and size of
immunoreactive proteins in total cellular protein as opposed to previous studies which only
probed cortical proteins. Furthermore, immunoblots were performed to test the specificity
of each antisera prepared against specific proteins in an attempt to clarify apparently
contradictory data obtained by previous studies using both polyclonal and monoclonal
antibodies.
Polyclonal antibodies against band A did not detect the presence of band A at 235
kDa, but did detect three bands between 100 and 140 kDa. Affinity-purified band C
antibodies were demonstrated to identify a 125 kDa protein from whole-cell lysates, and no
bands above 125 kDa, as previous studies have recognized. Interestingly, the 23 kDa
polyclonal antibody recognized not only a 23 kDa protein from the total cellular protein, but
a high molecular weight protein (above 125 kDa) as well.
