We have examined the interaction of exogenous 125I-discoidin I with living wild type (NC-4) Dictyostelium discoideum cells and the subcellular localization of endogenous discoidin I during development. The living cells bind 125I-discoidin I relatively rapidly, with an apparent steady state being reached within 40 min at 23°C. The rate of association is reduced to a similar extent by lowering the temperature to 4°C and by glutaraldehyde fixation of the cells. The level of steady-state binding is not developmentally regulated. Living cells do not degrade significant amounts of the 125I-discoidin I during incubations at 23°C. Differentiated cells bind the 125I-discoidin I via both the carbohydrate or C site receptors and the ionic or I site receptors, first identified on the corresponding glutaraldehyde-fixed cells (Bartles, J.R. and Frazier, W.A. (1982) Biochim. Biophys. Acta 687, 121-128). Depending on the concentration of 125I-discoidin I, 50% or more of the binding to the 8.5-h differentiated cells is via the I sites. Binding exhibits apparent positive cooperativity with respect to discoidin I. This causes the binding to appear nonsaturable, with a capacity for more than 107 discoidin I tetramers per cell. Independent verification of the nonsaturability of the interaction was obtained from experiments using diazotized [125I]iodosulfanilic acid to radiolabel the surfaces of cells treated with increasing concentrations of unlabeled discoidin I. The susceptibility of the surface radiolabeled discoidin I to various extractive treatments of the cellular particulate fraction suggests that as much as 10% is very tightly associated with cellular membranes. Dissociation experiments also suggest the existence of an irreversible component of binding, the magnitude of which increases with time of association. While the 8.5-h differentiated NC-4 cells exhibit maximal developmental cohesiveness and contain about 5·106 discoidin I tetramers per cell, cell surface labeling and radioimmunoassay indicate that they display only 1.3·103 tetramers per cell on their surface and only 6·103 tetramers per cell in their surrounding extracellular medium.
- (D. discoideum)
- Binding cooperativity
- Discoidin I-membrane interaction
ASJC Scopus subject areas
- Cell Biology