Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Functions of a-Tropomyosin Are Mainly Dependent upon the Local Structures of the Amino Terminus

a-Tropomyosin의 아미노 말단 구조가 기능에 미치는 영향

  • Published : 2004.10.01
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Abstract

It has been previously reported that unacetylated a-tropomyosin(TM) produced in E. coli failed to bind to actin while acetylated muscle TM and Ala-Ser dipeptide fusion TM (AS-TM) bound well to actin. In order to determine the structural requirement of the amino terminus for high actin affinity, a recombinant tropomyosin (Ala-TM) that a single Ala residue was added to the amino terminus of Ala-TM was constructed, overexpressed, and purified from E. coli. Actin affinity of Ala-TM was 2.3$\times$$10^{6}$$M^{-1}$, whereas that of unacetylated TM was considerably lower than 0.1$\times$$10^{-6}$$M^{-1}$ indicating that addition of a single Ala residue to the amino terminus drastically increased, at least twenty times, actin affinity of TM. Ala-TM, however, bound to actin about three times weaker than acetylated TM and AS- TM, implying that the addition of an Ala residue was insufficient for complete restoration of high actin affinity. While Ala-TM, AS-TM, and muscle TM showed inhibition and activation of actomyosin Sl ATPase activity depending on myosin Sl concentration, the degree of inhibition and activation was different from each other. AS-TM exhibited the greatest inhibition of the ATPase at low Sl concentration, whereas the greatest activation of the ATPase was observed with muscle TM. These results, together with previous findings, strongly suggested that local structure of the amino terminus is the crucial functional determinant of TM.

a-Tropomyosin (TM)의 아미노(N) 말단 구조의 중요성을 확인하기 위하여 N 말단에 알라닌 아미노산 잔기 하나를 첨가한 재조합 Ala-TM을 제조하였다. Ala-TM을 대장균에서 대량발현 시켜 정제한 후, N 말단이 아세틸화된 근육TM및 N말단에 알라닌-세린 잔기를 첨가한 AS-TM과 N말단이 비아세틸화된 TM등의 재조합 TM과 기능을 비교하였다. Ala-TM은 비아세틸화된 TM보다 액틴친화력이 현저히 증가했으나, 근육 및 AS-TM 보다는 약 3배정도 약하게 액틴에 결합하였다. 근육 TM, AS-TM,그리고 Ala-TM모두가 myosin 51의 농도가 낮을 때 ATPase 활성을 억제하였고 농도가 높을 때 촉진하였으나, 억제와 촉진의 정도는 서로 차이가 있었으며 비아세틸화된 TM은 억제하지 않았다. 이들 결과는 N말단 구조가 TM의 기능을 결정하는 중요한 요소임을 나타내며 TM의 온전한 기능을 위해서는 아세틸화된 N 말단이 필요하다는 것을 의미한다.

Keywords

References

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