한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제29권2호
  • /
  • Pages.90-95
  • /
  • 2001
  • /
  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

분광광도법에 의한 Cathepsin B 저해물질의 효소동력학적 저해특성 조사

Kinetic Analysis of Cathepsin B Inhibitor Using a Spectrophotometric Assay

  • 한길환 (영남대학교 응용미생물학과) ;
  • 김상달 (영남대학교 응용미생물학과)
  • 발행 : 2001.06.01
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초록

Cysteine 계 cathepsin B 단백질 분해 효소는 고등동물의 세포조직 내에 lysosome에 존재하며 병원성세균의 침입을 막고 불필요한 단백질을 분해시키며 또한 면역세포의 항원인식에 대한 단백질 생산 등에 관여하는 효소이다. 이 cathepsin B의 과량 발현은 암전이, 만성적 염증성 질환, 류머티스 관절염, 노인성치매 등의 원인이 된다. 이 cathepsin B를 저해하는 것으로 밝혀진 S. luteogriseus KT-10으로부터 분리한 저해제 KHS10을 이용하여 분광광도계를 사용, 그 저해력을 조사하였다. Cathepsin B 저해제 KHS10은 경쟁적 저해를 나타내며 cathepsin B는 기질 CLN에 대해 Km 값은 0.5mM을 Vmax 값은 $29.4\mu$M로 나타내었으며, 합성기질인 BANA에 대해서는 Km 값이 2mM을 Vmax 값은 7.8$\mu$M/min로 나타났다. 저해제 KHS10의 Ki 값은 $0.43{\mu}$M로 측정되었다. Cathepsin B에 대한 저해제 KS10의 반응시간에 대한 저해력은 1시간 반응시 저해력이 100%에 가깝게 높게 나타났으며 온도에 의한 저해율은 $25^{\circ}C$에서 활성이 가장 높게 나타났다. Cathepsin B와 저해제 KHS10이 반응 전 배양은 5분간의 전 배양으로 높은 저해율을 나타내었으며 또한 pH에 의한 저해활성은 pH 6.0에서 가장 높게 저해하는 것으로 조사되었다. 저해제 KHS10는 $100^{\circ}C$에서 1시간 열을 가해도 그 잔류 저해력은 80% 이상 유지되었다.

Kinetic Analysis of Cathepsin B Inhibitor Using a Spectrophotometric Assay. Han, Kil-Hwan and SangDal Kim*. Department of Applied MicrobioJ0f5Yt Yeungnam UniversitYt Kyongsan 77 2-749, Korea - The KHS 10, C4Hl10~6 formula produced from Streptomyces luteogriseus KT-] 0 effectively inhibited a lysosomal cysteine proteinase, cathepsin B. It inhibited the enzyme activity of cathepsin B competitively when the N a-CBZ-Llysine p-nitrophenyl ester HC] (CLN) was used as a substrate. The inhibition const:mt (Ki) of KHS 1 0 for cathepsin B detennined by spectrophotometeric assay was 430 nM. The effective inhibition of cathepsin B was observed at $25^{\circ}C$ :md pH 6.0. The cathepsin B inhibitor, KHSlO needed a preincubation of cathepsin B with the inhibitor for over 5 min. The KHS 10 preserved over 80% inhibition activity even after heat-treatment at $100^{\circ}C$ for ] hr.

키워드

참고문헌

  1. J. Antibiot. v.22 Leupeptins, new protease inhibitor isolated from Actinomycetes Aoyagi, T.;T. Takeuchi;A. Matsuzaki;Kawamura;K. S. Kondo;Hamada;M. K. Maeda;H. Umezawa
  2. Anal. Biochem. v.68 Specific spectrophotometric assays for cathepsin B. Bajkowski, A. S.;A. Frankfater
  3. Anal. Biochem. v.47 A new assay for cathepsin B and other thiol proteinases Barrett, A. J.
  4. Proteinase in mammalian cells and tissue Introduction to the history and classification of tissue proteinase Barrett, A. J.;A. J. Barrett(ed.)
  5. Biochem. Biophys. Res. Commun. v.120 The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors Barrett, A. J;M. E. Davies;A. Grubb.
  6. Annu. Rev. Physiol. v.59 Emerging roles for cysteine proteases in human Biology Chapman H. A.;R. J. Riese;G. P. Shi.
  7. Lysosomes in Biology and Pathology v.1 de Duve, C.;J. T. Dingle(ed.);H. B. Fell(ed.)
  8. Clin. Cancer Res. v.2 Protease as prognostic markers in cancer Duffy, M. J.
  9. Anticancer Res. v.14 Prognostic value of increased lung tumor tissue cathepsin B. Ebert, W.;H. Knoch;B. Werle;G. Trefz;T. Muley;E. Spiess
  10. J. Mol. Biol. v.234 Conformational variability of chicken cystatin: comparison of structures determined by X-ray diffraction and NMR spectroscopy Engh, R. A.;T. Dieckmann;W. Bode;E. A. Auerswald;V. Turk;R. Huber;H. Oschkinat
  11. J. Microbiol. Biotechnol. v.7 Selection and identification of a strain KT-10 producing the cathepsin B inhibitor Han, K. H.;S. D. Kim
  12. Kor. J. Appl. Microbiol. Biotechnol. v.29 Isolation and characterization of cathepsin B inhibitor produceded by Streptomyces luteogriseus KT-10 Han, K. H.;S. D. Kim
  13. FEBS Lett. v.231 Effect of proteinase inhibitors on intracellular processing of cathepsin B, H, and L in rat macrophages Hara, K.;E. Kominami;N. Katunuma
  14. Curr. Top. Cell. Regul. v.22 Structures and functions of lysosomal thiol proteinases and their endogenous inhibitor Katunuma, N.;E. Kominami
  15. Clin. Cancer Res. v.2 Cathepsin D, B, and L in malignant human lung tissue Ledakis, P.;T. T. William;N. Rosenberg;D. Romero-Fischmann;I. Daskal;T. T. Lah
  16. Curr. Top. Cell. Regul. v.17 Inhibitors associated with the proteinases of mammalian cells and tissues Lenney, J. F.
  17. Proc. Natl. Acad. Sci. USA v.73 Role of proteolytic enzymes in biological regulation Neurath, H.;K. A. Walsh
  18. Proc. Natl Acad. Sci. USA v.92 Percellular mobilization of the tissue destructive cysteine proteases, cathepsin B, L, and S by human macrophages Reddy U. Y.;Q-Y. Zhang;S. J. Weiss
  19. Cancer Metastasis Rev. v.9 Cathepsin B and its endogenous inhibitors: role in tumor malignancy Sloane, B. F.;K. Moin;E. Krepela;J. Rozhin
  20. Biochemical and Molecular Aspects of Selected Cancers v.2 Regulation of lysosomal endopeptidases in malignant neoplasia Sloane, B. F.;K. Moin;T. T. Lah;G. Pretlow(ed.);H. Pretlow(ed.)
  21. Biochem. J. v.316 Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4 guanidino) butane (E-64) is one of the most effective low M inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64- ß-trypsin complex Sreedharan, S. K.;C. Verma;L. S. Caves;S. M. Brocklehurst;S. E. Gharbia; H. N. Shah;K. Brocklehurst
  22. J. Antibiot. v.25 Antipain, a new protease inhibitor isolated from Actinomycetes Suda, H.;T. Aoyagi;M. Hamada;T. Takeuchi;H. Umezawa
  23. FEBS Lett. v.339 Kinetic of inhibition of bovine cathepsin S by bovine stefin B. Turk, B.;A. Colic;V. Stoka;V. Turk
  24. Agric. Biol. Chem. v.43 Purification and characterization of crystalline microbial alkaline proteinase inhibitors (MAPI)produced by Streptomyces nigrescens WT-27 Watanabe, T.;S. Murao