Interaction of Lys-61 labeled actin with myosin subfragment-1 and the regulatory proteins.
Actin modified at Lys-61 with fluorescein 5-isothiocyanate (FITC) recovers the ability to polymerize following the binding of phalloidin. The resulting polymer (FITC-P-actin) activates the S1-Mg2+-ATPase activity to the same extent as non-labeled F-actin. However, in the absence of phalloidin, FITC-actin (0.5 mg/ml) neither polymerized nor activated the S1-Mg2+-ATPase activity effectively even when it was preincubated with S1 for 3 h in 0.1 mM ATP, 0.1 mM CaCl2, and 1 mM Tris/HCl (pH 8.0), in contrast to the previous report [Miller, L., Phillips, M., & Reisler, E. (1988) Eur. J. Biochem. 174, 23-29]. The modification of Lys-61 did not impair the ability to bind tropomyosin or tropomyosin-troponin. On the other hand, the fluorescence polarization of FITC-P-actin increased when tropomyosin or troponin-tropomyosin was added. Moreover, the modification of Lys-61 affected the regulation of the actin activation of the S1-Mg2+-ATPase activity by the tropomyosin and troponin complex. In 30 mM KCl, 2.5 mM ATP, and 5 mM MgCl2, tropomyosin alone has been shown to inhibit the actin-activated S1-Mg2+-ATPase. This inhibition did not occur with FITC-P-actin even though tropomyosin was tightly bound. When troponin-tropomyosin was added, the FITC-P-actin activation of S1-Mg2+-ATPase activity was regulated in response to micromolar Ca2+ concentrations. On the other hand, in 30 mM KCl, 2.5 mM ATP, and 2 mM MgCl2, tropomyosin alone did not inhibit the actin-activated S1-Mg2+-ATPase activity with either non-labeled F-actin or FITC-actin.(ABSTRACT TRUNCATED AT 250 WORDS)
Miki
M
M
Department of Anatomy, University of Sydney, N.S.W., Australia.
D000199:Actins / Q000032:analysis*; D000251:Adenosine Triphosphatases / Q000032:analysis; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007202:Indicators and Reagents; D008239:Lysine / Q000032:analysis*; D015879:Myosin Subfragments / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013861:Thiocyanates; D014335:Tropomyosin / Q000032:analysis; D014336:Troponin / Q000032:analysis; D014783:Viscosity
D000199:Actins; D005452:Fluoresceins; D007202:Indicators and Reagents; D015879:Myosin Subfragments; D013861:Thiocyanates; D014335:Tropomyosin; D014336:Troponin; D000251:Adenosine Triphosphatases; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine