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Fig. 3. Interaction between -skeletal-muscle actin and the different mutants with thymosin ß4, profilin-IIA, DNase I and vitamin-D-binding protein (DBP). ³⁵S-Labeled -skeletal-muscle actin and mutants were produced by in vitro translation, run on native gels after mixing with thymosin ß4, profilin IIa, DNase I or DBP and subjected to autoradiography. Wild-type -skeletal-muscle actin shifted up (profilin, DNase I, DBP) or down (thymosin ß4) in complex with ABPs compared with -skeletal-muscle actin alone. Most of the mutants interacted with the different ABPs (Table 2). R183C and G15R are shown as examples of those that behaved like the wild type; the others are not shown but are listed in Table 2. The control lanes in each case represent the mutant actin run in the absence of added ABP. The mutant I64N was only partially shifted when DNAse I was added (asterisk). The mutants L94P and E259V remained associated with CCT (arrow) and prefoldin and the released product (arrowhead) showed little (L94P) or no (E259V) binding to ABPs. The mutants G182D and V163L, which stuck to CAP, did not shift with thymosin ß4 (up arrow), which might reflect the relatively low affinity of actin for thymosin and competition with CAP. Both mutants shifted with DBP and DNase I, however, indicating proper folding. The gel shown is a representative of three experiments with similar results.

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