Effects of pronase and concanavalin A upon the freeze-etch morphology of cell membranes of intact human erythrocytes

Abstract

Freeze-etch studies reveal that mild pronase treatment with subsequent incubation in concanavalin A induces aggregation of intramembranous particles (IMP) in intact human erythrocytes. This alteration in particle distribution is accompanied by a change in the distribution of the Con A molecules such that they also become clustered on the extracellular etch face. If divalent succinyl Con A is used after pronase instead of tetravalent Con A the IMPs still become clustered. Pronase only, Con A only, or succinyl Con A only does not cause the IMPs to become aggregated. Most surprising is the finding that pronase followed by Con A causes partial haemolysis of the cells whereas pronase only, Con A only, or pronase+succinyl Con A do not cause this haemoglobin loss. These perturbations of the erythrocyte plasma membrane appear to be a result of the pronase+Con A exerting a transmembrane effect on the spectrin. This conclusion is supported by sodium dodecylsulphate polyacrylamide gel electrophoresis of material crosslinked with dimethyl adipimidate dihydrochloride, which indicates that spectrin is more susceptible to being cross-linked after pronase+Con A; i.e. the spectrin is probably aggregated by the enzyme and lectin incubation.