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Biochemistry. 1990 Mar 27;29(12):3013-23.

The rigor configuration of smooth muscle heavy meromyosin trapped by a zero-length cross-linker.

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  • 1Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka, Japan.


When chicken gizzard heavy meromyosin (HMM) in its rigor complex with actin was reacted with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), HMM cross-linked with actin but also the two heads of the HMM molecule cross-linked to each other [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry 28, 1898-1904, 1905-1912]. By ultracentrifugal fractionation of the EDC-treated acto-HMM in the presence of Mg-ATP, we obtained a preparation enriched for gizzard HMM with cross-linked heads. When HMM molecules in this preparation were rotary-shadowed and observed in an electron microscope, many head pairs were in contact with each other. The amount of HMM with cross-linked heads determined by electron microscopy was equal to that of the cross-linked NH2-terminal 24K tryptic fragments of HMM heavy chains determined by NaDodSO4 gel electrophoresis, indicating that this cross-linking is primarily responsible for the contact observed between two HMM heads. Most pairs of the contacted heads originated in the same HMM molecule, although a few pairs belonged to different HMM molecules. Cross-linking between the two heads of the same HMM molecule appeared to occur within the distal, more globular half of each head. However, the cross-linking sites were located at different positions within the globular portion. The actin-activated Mg-ATPase activity of the HMM sample treated with EDC in the presence of actin increased in a biphasic manner, depending on the concentration of F-actin, with two apparent association constants: 2.9 x 10(4) M-1 and one much less than 1 x 10(4) M-1. Since the apparent association constant obtained with the HMM control was similar to the latter value, the association constant for HMM molecules with cross-linked heads was identified to be the former value. The binding of HMM to actin was thus strengthened at least by a factor of 3 by the cross-linking between two HMM heads. These results suggest that HMM heads are trapped by treatment with EDC in the rigor complex configuration and that this configuration is retained even after the HMM has been released from actin. The EDC reactivity of rabbit skeletal muscle HMM, however, was different from that of chicken gizzard HMM. The treatment of acto-HMM complexes with EDC did not generate cross-linking between two skeletal muscle HMM heads.

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