Dataset Information

Calcium-mediated regulation of recombinant hybrids of full-length Physarum myosin heavy chain with Physarum/scallop myosin light chains.

ABSTRACT: Physarum myosin is a Ca(2+)-binding protein and its activity is inhibited by Ca(2+) In the present study, to clarify the light chains (LCs) from the different species (Physarum and scallop) and to determine the specific Ca(2+)-regulated effects, we constructed hybrid myosins with a Physarum myosin heavy chain (Ph·HC) and Physarum and/or scallop myosin LCs, and examined Ca(2+)-mediated regulation of ATPases and motor activities. In these experiments, it was found that Ca(2+) inhibited motilities and ATPase activities of Physarum hybrid myosin with scallop regulatory light chain (ScRLC) and Physarum essential light chain (PhELC) but could not inhibit those of the Physarum hybrid myosin mutant Ph·HC/ScRLC/PhELC-3A which lacks Ca(2+)-binding ability, indicating that PhELC plays a critical role in Ca(2+)-mediated regulation of Physarum myosin. Furthermore, the effects of Ca(2+) on ATPase activities of Physarum myosin constructs are in the following order: Ph·HC/PhRLC/PhELC > Ph·HC/ScRLC/PhELC > Ph·HC/PhRLC/ScELC > Ph·HC/ScRLC/ScELC, suggesting that the presence of PhRLC and PhELC leads to the greatest Ca(2+) sensitivity of Physarum myosin. Although we did not observe the motilities of Physarum hybrid myosin Ph·HC/PhRLC/ScELC and Ph·HC/ScRLC/ScELC, our results suggest that Ca(2+)-binding to the PhELC may alter the flexibility of the regulatory domain and induce a 'closed' state, which may consequently prevent full activity and force generation.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC4913522 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Publications

Calcium-mediated regulation of recombinant hybrids of full-length Physarum myosin heavy chain with Physarum/scallop myosin light chains.

Zhang Ying Y Kawamichi Hozumi H Kohama Kazuhiro K Nakamura Akio A

Acta biochimica et biophysica Sinica 20160428 6

Physarum myosin is a Ca(2+)-binding protein and its activity is inhibited by Ca(2+) In the present study, to clarify the light chains (LCs) from the different species (Physarum and scallop) and to determine the specific Ca(2+)-regulated effects, we constructed hybrid myosins with a Physarum myosin heavy chain (Ph·HC) and Physarum and/or scallop myosin LCs, and examined Ca(2+)-mediated regulation of ATPases and motor activities. In these experiments, it was found that Ca(2+) inhibited motilities ...[more]

PMID: 27125976

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Project description:The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca(2+)-binding properties of the light chains. The Ca(2+)-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH(2) thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca(2+)-binding properties of light chains and generation of tension. When the SH(2) moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca(2+)-binding properties of light chain LC(2) is lost; under these conditions the Ca(2+)-binding affinity value of SH(2)-N-ethylmaleimide-blocked myosin (3.3x10(4)m(-1)) decreases to near that expressed with the dissociated light chain LC(2) (0.7x10(4)m(-1)). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH(2) thiol does not affect Ca(2+) binding. The native secondary and tertiary structure of myosin seem to be required for Ca(2+) binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH(2)-N-ethylmaleimide-blocked myosin normal Ca(2+)- and (Mg(2+)+actin)-stimulated ATPase activities are expressed; however, there is a loss in K(+)-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca(2+) with alterations in pH values. In the absence of Ca(2+)/EGTA buffer the biphasic Ca(2+)-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2x10(6)m(-1) and site two: 0.4x10(6)m(-1)) as compared with values obtained at pH6.5 (site one: 0.64x10(6)m(-1) and site two: 0.2x10(6)m(-1)). The Ca(2+)-binding affinity of light chain LC(2) and S(1), where the (S-1)-(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca(2+)-binding affinity, approx. 0.7x10(4)m(-1), whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca(2+)-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S(1) myosin subfragment light chain LC(2) was lost and thus was added back to the purified S(1) fraction. Light chain LC(2) was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)-(S-2) junction is needed for the positioning of light chain LC(2) and thus influences its essential conformation for Ca(2+) binding.

Dataset Information

Calcium-mediated regulation of recombinant hybrids of full-length Physarum myosin heavy chain with Physarum/scallop myosin light chains.

Publications

Calcium-mediated regulation of recombinant hybrids of full-length Physarum myosin heavy chain with Physarum/scallop myosin light chains.

Similar Datasets

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