한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제29권2호
  • /
  • Pages.84-89
  • /
  • 2001
  • /
  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Streptomyces luteogriseus KT-10 이 생산하는 Cathepsin B 저해물질의 분리 및 특성

Isolation and Characterization of Cathepsin B inhilbitor Produced by Streptomyces luteogriseus KT-10

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

포유동물조직의 세포내에 존재하는 lysosomal cysteine계 cathepsin B 효소는 암 발병과 전이, 류머티스 관절염, 염증 및 노인성치매 등의 여러 질병에 관여하고 있다. 이 cathepsin B를 저해하는 저해물질이 Streptomyces luteogriseus KT-10에 의해 생산되어 분리되었다. S. luteogriseus KT-10이 생산하는 cathepsin B 저해물질은 열에 안정하며 산, 알칼리에도 안정한 물질로 구성되어 있다. Cathepsin B 저해물질은 DEAE-Sephadex A-25, Sephadex G-15, silica gel, Sephadex LH-20의 column chromatography 과정을 거친 후 분취용 HPLC를 이용 분취한 후 백색분말의 cathepsin B 저해물질을 정제하였다. 이 정제한 저해물질은 UV 상의 전 범위에서 특이한 검출부위를 확인할 수 없었으며, $H_2$O, methanol, ethanol, n-butanol에는 녹지만 비극성인 chloroform, n-hexane, benzene 등에는 불용성이었다. 또한 ninhydrine, $H_2$$SO_4$, iodine에 양성 반응이지만 Ehrlichs, Pauly, Sakaguchi, phthalic acid, DNS, aniline 등의 단백반응과 당 반응에는 음성 반응으로 나타났다. IR 기기에서는 OH 기와 CH 기의 peak를 확인하였으며 $^1$H NMR 기기로 OH peak와 NH peak 또한 확인할 수 있었다. $^{13}$ C NMR spectrum은 4개의 탄소 peak를 가진 물질로 확인된 분자량이 207 dalton인 저해물질이다. 원소 분석기를 이용한 저해물질 분석 결과 분자식이 $C_4$$H_{11}$ $O_4$$N_{6}$로 나타났다. 이 cathepsin B 저해물질의 저해양식은 competitive 저해로 작용함을 확인할 수 있었다.

Isolation and Characterization of Cathepsin B inhibitor Produced by Streptomyces luteogriseus KT-IO. Han, Kil~Hwan and Sang~Dal Kim*. Department of Applied Microbiology, Yeungnam Universit}/t Kyongsan 712749, Korea - The cathepsin B inhibitor produced by Streptomyces luteogriseus KT-IO was very stable in heat, acidic and alkaline conditions. The cathepsin B inhibitor was isolated from the extracted fraction of culture broth with butanol, methanol and chloroform subsequently, the inhibitor was purified with following several column chromatography sLlch as DEAE-Sephadex A-25, Sephadex G-15, silica gel 60, Sephadex LH-20, and preparative HPLC. The cathepsin B inhibitor showed positively to detective reaction of ninhydrine, 5% H2S04, iodine, but negatively to the reaction of Ehrlich's reagent, DNS, aniline. The molecular formular of cathepsin B inhibitor was elucidated by JR, lH and 13C-NMR, FAB mass and elemental analyzer. Consequently, it was identified as C4HlI04N6. The cathepsin B inhibitor had the mode of competitive inhibition with the reaction of cathepsin B.

키워드

참고문헌

  1. J. Antibiot v.22 Leupeptins, new protease inhibitor isolated from actinomycetes Aoyagi, T.;T. Takeuchi;A. Matsuzaki;K. Kawamura;S. Kondo;M. Hamada;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. Biophysical 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.;W. T. Dieckmann;E. A. Bode;V. Auerswald;R. Turk;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. 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
  13. Curr. Top. Cell. Regul. v.22 Structures and Functions of Lysosomal Thiol Proteinases and Their Endogenous Inhibitor Katunuma, N.;E. Kominami
  14. 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
  15. Curr. Top. Cell. Regul. v.17 Inhibitors Associated with the Proteinases of Mammalian Cells and Tissues Lenney, J. F.
  16. Proc. Natl. Acad. Sci. USA v.73 Role of Proteolytic enzymes in biological regulation Neurath H;K. A. Walsh
  17. 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
  18. Cancer Metastasis Rev. v.9 Cathepsin B and its endogenous inhibitors: role in tumor malignancy Sloane, B. F;K. Moin;E. Krepela;J. Rozhin
  19. 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.)
  20. Biochem. J. v.316 Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane(E-64)is one of the most effective low Mr 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. D. Caves;S. M. Brocklehurst;S. E. Gharbia;H. N. Shah;K. Brocklehurst
  21. J. Antibiot v.25 Antipain, a new protease inhibitor isolated from Actin-omycetes Suda, H.;T. Aoyagi;M. Hamada;T. Takeuchi;H. Umezawa
  22. 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