Interaction of tropomyosin with F-actin-heavy meromyosin complex.
The effect of phosphorylated and dephosphorylated heavy meromyosins (HMMs) saturated with Ca2+ or Mg2+ on the binding of tropomyosin to F-actin and on the conformational changes of tropomyosin on actin was investigated. The experimental data were analysed on the basis of th emodel of cooperative binding of tropomyosin to F-actin with overlapping binding sites. In general, attachment of both HMMs to F-actin increased around 100-fold the tropomyosin-binding affinity but concomittantly reduced the cooperatively of binding. In the presence of Ca2+ and in the absence of ATP the binding of tropomyosin to F-actin in a "doubly contiguous" manner was three-fold stronger for F-actin saturated with dephosphorylated HMM as compared to phosphorylated HMM. Under the same rigor conditions but in the absence of Ca2+ the reverse was true but the difference was about 1.5-fold. The binding stoichiometry of tropomyosin to actin was 7:1 in the presence of dephosphorylated HMM saturated with Ca2+ or phosphorylated-saturated with Mg2+ and tended to be about 6:1 for both after the exchange of the cation bound to myosin heads. Bound HMM was also found to influence the fluorescence polarization of 1,5-IAEDANS-labelled tropomyosin complexed with F-actin in muscle ghost fibres. In the presence of Ca2+, the amount of randomly arranged tropomyosin fluorophores decreased when dephosphorylated HMM was bound to ghost fibres, in contrast to an observed increase in the case of bound phosphorylated HMM. Thus HMM induced conformational changes of tropomyosin in the actin-tropomyosin complex that was reflected in an alteration of the geometrical arrangement between tropomyosin and actin.(ABSTRACT TRUNCATED AT 250 WORDS)
Szczesna
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D
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
Borovikov
Y S
YS
Kakol
I
I
Sobieszek
A
A
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D005454:Fluorescence Polarization; D007457:Iodine Radioisotopes; D008274:Magnesium / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000378:metabolism*
D000199:Actins; D007457:Iodine Radioisotopes; D015879:Myosin Subfragments; D014335:Tropomyosin; D008274:Magnesium; D002118:Calcium