Interaction of Lys-61 Labeled Actin with Myosin Subfragment-1 and the Regulatory Proteins1

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Abstract
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 polmerized nor activated the Sl-Mg2+-ATPase activity effectively even when it was preincubated with SI for 3 h in 0.1 mM ATP, 0.1 mM CaCl2, and 1 mM Tris/HCl (pH8.0), in contrast to the previous report [Miller, L., Phillips, M., & Reisler, E. (1988) Eur. J. Biochenu 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 Sl-Mg2+-ATPase activity by the tropomyosin and troponin complex. In 30 mM KC1, 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 Sl-Mg2+-ATPase activity was regulated in response to micromolar Ca2+ concentrations. On the othr hand, in 30 mM KC1, 2.5 mM ATP, and 2 mM MgCl2, tropomyosin alone did not inhibit the actin-activated Sl-Mg2+-ATPase activity with either non-labeled F-actin or FITC-actin. When troponin-tropomyosin was added, the ATPase activity was always inhibited to a certain extent with FITC-P-actin whether Ca2+ was present or not, although a strong inhibition in the absence of Ca2+ and no inhibition in the presence of Ca2+ were observed with non-labeled F-actin. These results indicate that Lys-61 is located in a region which is closely related to the regulation of the actin-myosin interaction by tropomyosin-troponin.