[2] Purification of myosin I and myosin I heavy chain kinase from Acanthamoeba castellanii
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Cited by (26)
Chapter 6 Kinetic and Equilibrium Analysis of the Myosin ATPase
2009, Methods in EnzymologyCitation Excerpt :These assays measure nonphysiological ATPase activities but are useful for determining the number of active myosins in a preparation and for measuring relative myosin concentrations in binding assays (e.g., section 4). The following procedure detects the steady‐state hydrolysis of radioactive ATP and has the advantage of being insensitive to phosphate‐contaminated myosin samples (Lynch et al., 1991). Nonradioactive methods for measuring phosphate are also available (Pollard, 1982), including a commercially available detection methods (Lanzetta et al., 1979; Webb, 1992).
Acanthamoeba myosin IC colocalizes with phosphatidylinositol 4,5-bisphosphate at the plasma membrane due to the high concentration of negative charge
2008, Journal of Biological ChemistryCitation Excerpt :Binding Assay—Proteins (40-100 nm) and vesicles were mixed together in 20 mm imidazole, pH 7.0, 100 mm NaCl, and 1 mm EGTA containing 0.5 mg/ml bovine serum albumin to block nonspecific binding. The mixture was centrifuged for 40 min at 200,000 × g. Aliquots of the supernatants were removed, and the amount of unbound FL-AMIC or T4 was determined by measuring K-ATPase activity (28). Vesicle concentration and pelleted vesicles were monitored by fluorescence.
Identification and characterization of an 8-kDa light chain associated with Dictyostelium discoideum MyoB, a class I myosin
2006, Journal of Biological ChemistryCitation Excerpt :This relationship holds for MyoD/AMIC, since AMIC has a 16.8-kDa CaM-like light chain (MICLC) that shares 43% sequence identity with MlcD (14, 38). On the other hand, AMIB co-purifies with one subunit of a 27-kDa protein and variable, and always less than equimolar amounts, of a 14-kDa protein (39, 40). The discrepancy in the size and nature of the light chains associated with AMIB and MyoB is surprising, especially in light of the fact that they have IQ motifs that share 61% sequence identity.
Myosin I Phosphorylation Is Increased by Chemotactic Stimulation
2001, Journal of Biological ChemistryCitation Excerpt :No 125-kDa band was visible either in the immunoblot or in the autoradiogram (Fig. 1 B, lane 2), again demonstrating the specificity of the antibody and the immunoprecipitation protocol. The MIHCKs from Acanthamoeba and Dictyostelium are present in high speed supernatants (29, 37), suggesting that a significant proportion of these enzymes may be readily solubilized. Since the myoB heavy chain has been shown to be a poor substrate for the Dictyostelium MIHCK (29), an extract was tested for a kinase activity that might phosphorylate the myoB heavy chain.
[5] Purification and assay of the Arp2/3 complex from Acanthamoeba castellanii
1998, Methods in Enzymology