Abstract
Using glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase as a linked enzyme assay for determination of free inorganic phosphate, as described by Trentham et al . (1972, Biochem. J. 126 , 635–644 ) we have been able to monitor the time course of P i release from F-actin following ATP hydrolysis that accompanies ATP-actin polymerization. The rate constant for P i dissociation from Mg-F-actin is 0.006 s −1 at 25°C and pH 7.8, both in the presence of 1 mM Mg and 0.1 M KC1 + 1 mM Mg. This result confirms the existence of ADP-P i-F-actin as a major intermediate in the polymerization of ATP-actin ( Carlier and Pantaloni, 1986, Biochemistry 25 , 7789–7792 ). The method is potentially useful for other enzymes hydrolyzing triphosphate nucleotides, provided that the rate of P i release is appreciably lower than 0.1 s −1.
Published Version
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