Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins.

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Abstract
The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. Rabbit antisera was produced against isolated ciliary membranes and against partially purified immobilization antigens derived from 3 serotypes (A, B and H). These antisera were used as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross-reactivities of the antisera were examined. The antigens recognized by these sera were identified by using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), .apprx. 250,000 MW. Several secondary antigens, including a family of polypeptides of 42,000-45,000 MW, are common to the membranes of serotypes A, B and H; antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for the studies on the effects of the antibodies on electrophysiological properties of cells and EM localization studies. These antibodies also were used to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i-antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. Antigen-antibody interaction per se is not sufficient for immobilization, antibody bivalency, which allows antigen cross-linking, is essential.