Polymerization of actin modified with fluorescein isothiocyanate.
Solution properties of skeletal muscle actin, modified at lysine-61 with fluorescein isothiocyanate (FITC) [Burtnick, L.D. (1984) Biochim. Biophys. Acta 791, 57-62], were re-examined in this work by light scattering, analytical ultracentrifugation, fluorescence, electron microscopy and myosin ATPase activity measurements. Fluorescence measurements using trace amounts of actin labeled with N-(1-pyrenyl)iodoacetamide showed that the FITC modification inhibited but did not block completely the polymerization of actin by KCl and MgCl2. Sedimentation velocity runs of FITC-actin, incubated with 100 mM KCl and 2 mM MgCl2, revealed the presence in these solutions of polymeric, oligomeric and monomeric species. The critical concentration for FITC-actin polymerization under these conditions was 12 microM. As judged by electron microscopy, FITC-actin polymers were similar to but generally shorter than standard F-actin filaments. Light scattering measurements indicated that FITC modification inhibited also the polymerization of actin by myosin subfragment 1 (S1) but the resulting complexes were indistinguishable from standard, decorated actin filaments. MgATPase measurements showed that FITC-actin, polymerized by preincubation with S1, activated the MgATPase activity of S1 while the monomeric labeled protein did not. Thus, in analogy to native actin, the activating function of FITC-actin depended on the formation of actin filaments. Results presented in this study suggest that the region around lysine-61 of actin plays an important role in actin-actin contact and is less crucial to actomyosin interaction.
Miller
L
L
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Phillips
M
M
Reisler
E
E
D000199:Actins / Q000032:analysis*; D000251:Adenosine Triphosphatases / Q000032:analysis; D000345:Affinity Labels; D000818:Animals; D001704:Biopolymers / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D008027:Light; D008239:Lysine / Q000032:analysis / Q000502:physiology; D046911:Macromolecular Substances / Q000032:analysis*; D008854:Microscopy, Electron; D009132:Muscles / Q000032:analysis / Q000201:enzymology; D011817:Rabbits; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates; D014461:Ultracentrifugation
D000199:Actins; D000345:Affinity Labels; D001704:Biopolymers; D005452:Fluoresceins; D046911:Macromolecular Substances; D013861:Thiocyanates; D000251:Adenosine Triphosphatases; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine