The effect of toxins on inorganic phosphate release during actin polymerization

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P i release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P i release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P i from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P i . The effect of both toxins can be sufficiently explained by local and mechanical blockade of P i dissociation.