Biochemical Studies on the Mode of Action of Cytochalasin B

Cytochalasin B binds rapidly and reversibly to human red blood cells.Scatchard plot analysis of binding data reveals a class of high a5ity binding sites (dissociation constant ~10~' M; 3 X lo5 sites per cell) and a class of low affinity binding sites (dissociation constant B10-5 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 a5ity 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-Glucase inhibits the high affinity binding by 80 to QO%, 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 aiIinity for the high affinity binding sites, whereas dihydrocytochaiasin B and the y-lactone derivative have a much lower affinity.Selective elution of spectrin and components 5 and 6 has no effect on the high a5ity binding of cytochalasin B to the membrane.Cytochalasin B, at low concentrations (lo-' M), inhibits hexose transport in several types of mammalian cells including red blood cells (l-6).At higher concentrations (10B6 to 10e4 M), it inhibits a wide variety of cellular movements (7).The mode of action of cytochalasin B at the molecular level is unknown, although there is evidence suggesting that the latter effect is not the result