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Journal of Muscle Research & Cell Motility

, Volume 11, Issue 4, pp 313–322 | Cite as

Covalent crosslinking of myosin subfragment-1 and heavy meromyosin to actin at various molar ratios: Different correlations between ATPase activity and crosslinking extent

  • Yi-Ping Huang
  • Michio Kimura
  • Katsuhisa Tawada
Papers

Summary

This paper describes a systematic study of crosslinking of skeletal muscle myosin subfragment-1 (S1) and heavy meromyosin (HMM) to F-actin in the rigor state with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). We followed the time courses of S1 or HMM head crosslinking at various actin:S1 or actin:HMM head molar ratios and the resulting superactivation of ATPase activity. The ATPase activity of the covalent complexes was measured at 0.5 M KCl, where the covalent complexes retain superactivated ATPase activity but the activity of uncrosslinked myosin heads is not activated by actin. S1 crosslinking was slowest at the actin:S1 molar ratio of 1∶1, but faster at larger molar ratios, where more than 80% of added S1 could be crosslinked to actin. In spite of the dependence of crosslinking rate on actin∶S1 ratio, there were two linear correlations between ATPase activity and the extent of S1 crosslinking to actin: one for S1 crosslinked to actin at actin∶S1 molar ratios more than 2.7∶1 and the other for S1 crosslinked at a molar ratio of 1∶1. Extrapolation of the former correlation line to 100% crosslinked S1 gave an ATPase activity of 39 s−1 for actin-S1 covalent complex at 25 °C, whereas that of the other correlation line gave 21 s−1. The latter smaller activity suggests that the interface between actin and S1 in their rigor complexes at a molar ratio of 1∶1 is different from that at molar ratios of more than 2.7∶1. The acto-HMM crosslinking rate depended on the ratio of actin to HMM head, like that of S1 crosslinking to actin. The ATPase activity of crosslinked actin-HMM was, unlike that of actin-S1 covalent complexes, bell-shaped as a function of the crosslinked heads, but chymotryptic conversion of HMM to S1 in the covalent complexes made the bell-shaped characteristics disappear and increased the activity close to that of actin-Sl covalent complexes. These results indicate that some physical constraint imposed on myosin heads suppresses the actin-activated ATPase activity of HMM crosslinked to actin.

Keywords

ATPase Activity Propyl Dimethylamino Carbodiimide Myosin Head 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Chapman and Hall Ltd 1990

Authors and Affiliations

  • Yi-Ping Huang
    • 1
  • Michio Kimura
    • 1
  • Katsuhisa Tawada
    • 1
  1. 1.Department of Biology, Faculty of ScienceKyushu UniversityFukuokaJapan

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