Biochemical Studies on the Mode of Action of Cytochalasin B

Abstract

Cytochalasin B binds rapidly and reversibly to human red blood cells. Scatchard plot analysis of binding data reveals a class of high affinity binding sites (dissociation constant ≅10^-7 m; 3 x 10⁵ sites per cell) and a class of low affinity binding sites (dissociation constant ≧10^-5 m). All of the high affinity binding sites are in the plasma membrane (i.e. ghost). Variation of ionic strength and of pH in the range of 5.0 to 9.0 has only a small effect on the ability of the membrane to bind cytochalasin B with high affinity. The high affinity binding is not affected when intact cells are treated with pronase or with trypsin, but is lost when ghosts are similarly treated under conditions where the inner surface of the membrane is accessible to these enzymes. This binding decreases by more than 90% when either intact cells or ghosts are treated with p-chloromercuribenzoate. d-Glucose inhibits the high affinity binding by 80 to 90%, whereas l-glucose has very little effect. The effectiveness of eight different sugars to inhibit high affinity binding of cytochalasin B to ghosts is related to their affinity for the sugar transport system. All of these results indicate that at least 80% of the high affinity cytochalasin B binding sites are intimately related to this transport system. As judged by competition experiments, cytochalasin A and B have the same affinity for the high affinity binding sites, whereas dihydrocytochalasin B and the γ-lactone derivative have a much lower affinity. Selective elution of spectrin and components 5 and 6 has no effect on the high affinity binding of cytochalasin B to the membrane.