| Kinase Assay: |
| Journal of biological chemistry, 1981, 256(3):1290. | | The effects of cytochalasins on lymphocytes. Identification of distinct cytochalasin-binding sites in relation to mitogenic response and hexose transport.[Reference: WebLink] |
METHODS AND RESULTS:
Cytochalasin B inhibits phytomitogen-induced human lymphocyte proliferation with a Ki of approximately 6 X 10(-6) M. Cytochalasin A, Cytochalasin C, Cytochalasin D, Cytochalasin E, and Cytochalasin H are also inhibitory with varying degrees of potency, whereas cytochalasin G and chaetoglobosins A, B, C, E, F, and J are not at concentrations as high as 15 microM. Cytochalasin B also competitively inhibits carrier-mediated equilibrium exchange of hexose (Ki of approximately 7 X 10(-7) M), but cytochalasin E is ineffective. Cytochalasin B binds reversibly to the lymphocyte at three distinct sites: L, M, and H. The ligand binding at L site shows the apparent dissociation constant (Kd) of 1 to 3 X 10(-6) M and total binding sites (Bt) of 6 to 8 X 10(7)/cell, represents approximately 85% of the total saturable binding, displays a broad specificity interacting with Cytochalasin C, cytochalasin D, and cytochalasin E, is not displaceable by D-glucose, is located mostly in a cytosol fraction, and exists in intimate relation to cytoskeletal actin. M site shows a Kd of 2 to 4 X 10(-7) M and Bt of 5 to 8 X 10(6)/cell, represents about 8% of the total saturable binding, shows stringent specificity not being displaced by Cytochalasin C, cytochalasin D, and cytochalasin E, is competitively displaced by D-glucose and phloretin, and is quantitatively recoverable in the plasma membrane fraction.
The binding to H site shows a Kd of 0.5 to 1.0 X 10(-7) M and Bt of 4 to 5 X 10(6)/cell, representing approximately 7% of the total saturable binding, shows a broad specificity, is insensitive to D-glucose, and is membrane bound.
CONCLUSIONS:
It is proposed that L site is actin and is involved in the inhibition of lymphocyte mitogenesis, whereas M site is associated with the hexose transport carrier. Structure-activity relationships of cytochalasin effects are also discussed. |
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