The effect of toxins on inorganic phosphate release during actin polymerization
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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.
KeywordsActin Phalloidin Jasplakinolide Inorganic phosphate Kinetics
This work was supported by the Hungarian Science Foundation (OTKA grant K77840 to M. Ny.) the Hungarian National Office for Research and Technology (GVOP-3.2.1.-2004-04-0190/3.0, and GVOP-3.2.1.-2004-04-0228/3.0) and the Science, Please! Research Team on Innovation (SROP-4.2.2/08/1/2008-0011) program. Miklós Nyitrai holds a Wellcome Trust International Senior Research Fellowship in Biomedical Sciences.
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