We have studied the kinetics of the gelation process that occurs upon warming cold extracts of Acanthamoeba using a low-shear falling ball assay. We find that the reaction has at least two steps, requires 0.5 mM ATP and 1.5 mM MgCl2, and is inhibited by micromolar Ca++. The optimum pH is 7.0 and temperature, 25 degrees-30 degrees C. The rate of the reaction is increased by cold preincubation with both MgCl2 and ATP. Nonhydrolyzable analogues of ATP will not substitute for ATP either in this "potentiation reaction" or in the gelation process. Either of two purified or any one of four partially purified Acanthamoeba proteins will cross-link purified actin to form a gel, but none can account for the dependence of the reaction in the crude extract on Mg-ATP or its regulation by Ca++. This suggests that the extract contains, in addition to actin-cross-linking proteins, factors dependent on Mg-ATP and Ca++ that regulate the gelation process.
Article| May 01 1980
Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors.
S D MacLean-Fletcher
T D Pollard
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1980) 85 (2): 414–428.
S D MacLean-Fletcher, T D Pollard; Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors.. J Cell Biol 1 May 1980; 85 (2): 414–428. doi: https://doi.org/10.1083/jcb.85.2.414
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