Biochemical studies on the mode of action of cytochalasin B. Preparation of (3H)cytochalasin B and studies on its binding of cells.

Tritium-labeled cytochalasin B of high specific activity (6 Ci per mmole) has been prepared by reduction of cytochalasin A with [3H]NaBH4. The structure of the radioactive product was shown to be identical with that of authentic cytochalasin B by high resolution mass spectrometry, chromatographic analysis, and derivatization. The synthesized compound produced the same effects as authentic cytochalasin B in a number of biological systems. The [3H]cytochalasin B was found to bind rapidly and reversibly to HeLa cells and to bovine blood platelets at both high (10-j M) and low (lop7 M) drug concentrations. Scatchard plots of binding data indicated that there is a class of high affinity binding sites with dissociation constants of about lo-’ M (about lo4 sites per platelet and lo6 sites per HeLa cell) and a class of low affinity binding sites with dissociation constants of about 10m5 M. Similar results were obtained with red blood cells and SV40 transformed mouse fibroblasts. Low affinity sites were also found in Dicfyosfelium discoideum amoebae, but high affinity sites were not detected. In comparison, bacterial cells bound relatively low levels of cytochalasin B at all drug concentrations. is accompanied by disruption or disappearance of microfilaments (3) which, in some cases, have been shown to be similar to actin filaments from muscle (9, and for review, see Reference 10). Indeed, cytochalasin I3 causes a decrease in the intrinsic viscosity of actin (ll), and, as judged by electron microscopy, the drug alters the morphology of actin filaments isolated from muscle and blood platelets (12). Thus, in some cases actin-like microfilaments of the cell may be the cytochalasin B receptor. However, such evidence is indirect, and it is possible that cytochalasin B inhibits movements by acting on some other component of the cell. The recent observations that cytochalasin B inhibits hesose transport (4-6) suggest that the cell membrane contains receptor(s) for the drug, and Bluemink (13), Estensen et al. (14), and Krishan (15) have suggested that the effects of the drug in general may be caused by its interaction with the plasma membrane. In order to identify and to characterize the cellular receptors of the drug, we have developed a facile procedure for preparing [3H]cytochalasin B of high specific activity. We report here initial studies on the binding of this [3H]cytochalasin B to a variety of cell types.