title: Measurement of Pi dissociation from actin filaments following ATP hydrolysis using a linked enzyme assay.
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 Pi release from F-actin following ATP hydrolysis that accompanies ATP-actin polymerization. The rate constant for Pi dissociation from Mg-F-actin is 0.006 s-1 at 25 degrees C and pH 7.8, both in the presence of 1 mM Mg and 0.1 M KCl + 1 mM Mg. This result confirms the existence of ADP-Pi-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 Pi release is appreciably lower than 0.1 s-1.
mesh_terms: D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D010710:Phosphates / Q000032:analysis / Q000378:metabolism*; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000199:Actins; D010710:Phosphates; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase