생명과학회지 (Journal of Life Science)

  • 제15권6호
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  • Pages.935-940
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  • 2005
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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형광단이 붙어 있는 인산결합 단백질에 의한 인산 배출의 실시간 측정

Real Time Scale Measurement of Inorganic Phosphate Release by Fluorophore Labeled Phosphate Binding Protein

  • 정용주 (국민대학교 생명나노화학과)
  • Jeong Yong-Joo (Department of Bio and Nanochemistry, Kookmin University)
  • 발행 : 2005.12.01
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초록

Coumarine이 부착된 인산결합 단백질 (PBP-MDCC)의 형광변화가 뉴클레오사이드 삼인산 가수분해과정에서 배출된 무기 인산의 양을 측정하기 위해 관찰되었다. PBP-MDCC 정제후, 형광 방출 스펙트럼은 형광세기가 PBP-MDCC의 몰비을로 약 $70\%$까지 직선형태로 증가하는 것을 보였다. 형광 신호와 인산 기준물질과의 상호관계 측정이 인산 농도-형광세기 표준곡선을 구하기 위하여 stopped-flow 기구에서 행하여졌다. dTTP 가수분해로 부터 나오는 에너지를 이용하여 이중나선 DNA를 풀어주는 단백질인 T7박테리오파지 나선효소를 dPTT라 반응 시켰을 때, 형광변화를 배출된 인산의 양으로 전환할 수 있었다. 인산 배출 결과는 단일가닥 Ml3 DNA가 T7나선 효소에 의한 dTTP가수분해반응을 여러배 증가시키는 것을 보인다. 뉴클레오타이드 삼인산 가수분해 반응에 있어서 종말점 분석 대신에, PBP-MDCC에 의한 연속적인 인산 배출 분석이 배출된 인산을 측정하는데 있어서 쉽고 편리한 방법임을 보였다.

Fluorescence change of coumarin labeled phosphate binding protein (PBP-MDCC) was monitored to measure the amount of released inorganic phosphate ($P_{i}$) during nucleoside triphosphate (NTP) hydrolysis reaction. After purification of PBP-MDCC, fluorescence emission spectra showed that fluorescence responded linearly to $P_{i}$ up to about 0.7 molar ratio of $P_{i}$ to protein. The correlation of fluorescence signal and $P_{i}$ standard was measured to obtain [$P_{i}$] - fluorescence intensity standard curve on the stopped-flow instrument. When T7 bacteriophage helicase, double-stranded DNA unwinding enzyme using dTTP hydrolysis as an energy source, reacted with dTTP, the change of fluorescence was able to be converted to the amount of released $P_{i}$ by the $P_{i}$ standard curve. $P_{i}$ release results showed that single-stranded Ml3 DNA stimulated dTTP hydrolysis reaction several folds by T7 helicase. Instead of end point assay in NTP hydrolysis reaction, real time $P_{i}$-release assay by PBP-MDCC was proven to be very easy and convenient to measure released $P_{i}$.

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참고문헌

  1. Baird, C. L., Gordon, M. S., Andrenyak, D. M., Marecek, J. F. and Lindsley, J. E. 2001. The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release. J. BioI. Chem. 276, 27893-27898 https://doi.org/10.1074/jbc.M102544200
  2. Banik, U. and Roy, S. 1990. A continuous fluorimetric assay for ATPase activity. Biochem. J. 266, 611-614
  3. Brune, M., Hunter, J. L., Corrie, J. E. and Webb, M. R 1994. Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33, 8262-8271 https://doi.org/10.1021/bi00193a013
  4. Brune, M., Hunter, J. L., Howell, S.A., Martin, S. R, Hazlett, T. L., Corrie, J. E. and Webb, M. R 1998. Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Biochemistry 37, 10370-10380 https://doi.org/10.1021/bi9804277
  5. De Groot, H. and Noll, T. 1985. Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions. Biochem. J. 230, 255-260
  6. Hibberd, M. G., Dantzig, J. A., Trentham, D. R and Goldman, Y. E. 1985. Phosphate release and force generation in skeletal muscle fibers. Science 228, 1317-1319 https://doi.org/10.1126/science.3159090
  7. Hibberd, M. G. and Trentham, D. R 1986. Relationships between chemical and mechanical events during muscular contraction. Annu. Rev. Biaphys. Biaphys. Chem. 15, 119-161 https://doi.org/10.1146/annurev.bb.15.060186.001003
  8. Hingorani, M. M. and Patel, S. S. 1996. Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases. Biochemistry 35, 2218-2228 https://doi.org/10.1021/bi9521497
  9. Hingorani, M. M., Washington, M. T., Moore, K. C. and Patel, S. S. 1997. The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase. Proc. Natl. Acad. Sci. USA 94, 5012-5017
  10. Hirshberg, M., Henrick, K., Haire, L. L., Vasisht, N., Brune, M., Corrie, J. E. and Webb, M. R. 1998. Crystal structure of phosphate binding protein labeled with a coumarin fluorophore, a probe for inorganic phosphate. Biochemistry 37, 10381-10385 https://doi.org/10.1021/bi980428z
  11. Ledvina, P. S., Yao, N., Choudhary, A. and Quiocho,F. A. 1996. Negative electrostatic surface potential of protein sites specific for anionic ligands. Proc. Natl. Acad. Sci. USA 93, 6786-6791
  12. Luecke, H. and Quiocho, F. A. 1990. High specificity of a phosphate transport protein determined by hydrogen bonds. Nature 347, 402-406 https://doi.org/10.1038/347402a0
  13. Matson, S. W. and Kaiser-Rogers, K. A. 1990. DNA helicases. [Review] [306 refs]. Annual Review of Biochemistry 59, 289-329 https://doi.org/10.1146/annurev.bi.59.070190.001445
  14. Patel, S. S. and Picha, K. M. 2000. Structure and Function of HexamericHelicases. Annual Review of Biochemistry 69. 651-697 https://doi.org/10.1146/annurev.biochem.69.1.651
  15. Patel, S. S., Rosenberg, A. H., Studier, F. W. and Johnson, K. A. 1992. Latge scale purification and biochemical characterization of T7 prirnase/helicase proteins. Evidence for homodimer and heterodimer formation. J. BioI. Chem. 267, 15013-15021
  16. Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W. and Patel, S. S. 1996. Biochemical Analysis of Mutant T7 Primase/Helicase Proteins Defective in DNA Binding, Nucleotide Hydrolysis, and the Coupling of Hydrolysis with DNA Unwinding. J. BioI. Chem. 271, 26825-26834 https://doi.org/10.1074/jbc.271.43.26825
  17. Yao, N., Ledvina, P. S.,Choudhary, A. and Quiocho, F. A. 1996. Modulation of a salt link does not affect binding of phosphate to its specific active transport receptor. Biochemistry 35, 2079-2085 https://doi.org/10.1021/bi952686r