DOI QR코드

DOI QR Code

택사(Alismatis Rhizoma) trypsin inhibitor의 정제와 특성

Purification and Characterization of Trypsin Inhibitor from Alismatis Rhizoma

  • 발행 : 2002.04.01

초록

한방재료의 하나인 택사(Alismatis Rhizoma, AR)로부터 단백성 trypsin inhibitor(TI)를 분리, 정제하여 특성을 조사하였다. 정제과정은 0-80.% 포화 황산암모늄을 이용한 염석법, DEAE-cellulose ion exchange chromatography, Sep-hadex G-150 chromatography 등을 이용하였다. 정제된 ARTI의 분자량을 gel filtration과 SDS-PAGE 한 결과 모두 약 23,000 Da으로 나타나 monomer로 되어 있는 것으로 나타났다. 온도안정성에 있어 0-6$0^{\circ}C$에서는 안정하였으나 그 이상의 온도에서는 약 35%가지 안정성이 떨어졌다. ARTI와 상품화된 soybean kunitz inhibitor의 저해능을 비교해 본 결과 ARTI 및 soybean inhibitor 각각의 농도가 0.071 $\mu$M, 1.7 $\mu$M일 때 0.025 g/$m\ell$ trypsin활성을 50% 정도 저해하는 것으로 나타났다. ARTI의 trypsin의 가수분해반응에 대한 저해형태는 비경쟁적 저해형인 것으로 나타났으며 km값은 0.81 $\mu$M이었다.

A trypsin inhibitor was isolated and purified from Azismatis Rhizoma which has been used as a galenic for diuretic and antiphlogistic. Purification was carried out by 0-80% saturated ammonium sulfate salting out, DEAE- cellulose ion exchange chromatogrphy, Sephadex G-150 gel filtration. The molecular weight of Alismatis Rhizoma trypsin inhibitor(ARTI) was estimated to be about 23,000 Da by gel filtration and SDS-PAGE, it must be monomer. ARTI was stable at 0~6$0^{\circ}C$, but at higher temperature its activity was decreased about 35%. When benzoyl-dl-arginine p-nitroanilide was used as a substrate of trypsin, half-maximal inhibition of ARTI was observed at 0.071 $\mu$M. ARTI inhibited the hydrolysis of trypsin non-competitively and Km value was 0.81 $\mu$M.

키워드

참고문헌

  1. Biochem. J. v.96 Teh gel filtration behaviour of proteins related to their molecular weight over a wide range Andrews;P. https://doi.org/10.1042/bj0960595
  2. J. Med. Chem. v.12 Irreversible enzyme I inhibitors CXLIV. Proteolytic enzymes VⅢ. Additional active site directed irreversible inhibitors of trypsin derived from m- and p- (phenoxyalkoxy)- benzamidines with a terminal sulfonyl fluoride Baker;B.R.;E.H.Ericksor https://doi.org/10.1021/jm00301a029
  3. Method Enzymol. v.11 Organophorus compounds Cohen;J.A;R.A.Oosterbaan;F.Berends https://doi.org/10.1016/S0076-6879(67)11085-9
  4. Acad. Sci. v.121 Disc electrophorsis-Ⅱ. Method and application to human serum proteins Davis;H.J.;N.Y.Ann https://doi.org/10.1111/j.1749-6632.1964.tb14213.x
  5. General Review v.55 Proteinase and their inhibitors;in Methods of Enzymatic Analysis Fritz;H;V.Seemuller;H. Tscheche.
  6. J. Ann. Chem. Soc. v.106 3-Alkoxy-7amino-4-chloroissocoumarins;a new class of suicide substrates for serine proteases Harper;J.W.;J.C.Powers https://doi.org/10.1021/ja00336a050
  7. J. Biol. Chem. v.179 Inhibition of the proteinase and esterase activition of trypsin and chymotrypsin by diisopropyl fluorophosphate. Crystallization of the inhibited chymotrypsin Jansin;E.J;M.D.F.Nutting;R.Jang;A.K.Ball
  8. Methods in enzymology part v.B45 Naturally occurring inhibitors of proteolytic enzymes Kassell;B.
  9. J. Gen. Physiol. v.17 Inactivation of crystalline trypsin Kunitz;M.;J.H.Northrop. https://doi.org/10.1085/jgp.17.4.591
  10. Nature v.T4 no.227 Cleavage of structural proteins during the assembly of the head of bacteriophage Laemmli;U.K. https://doi.org/10.1038/227680a0
  11. Ann. Rev. Biochem. v.49 Protein inhibitors of proteinases Laskowaski;M.J.;I.A.Kato https://doi.org/10.1146/annurev.bi.49.070180.003113
  12. J. Biochem. v.193 Protein measurement with the folin phenol reagent Lowry;S.O.N.J.Resebrough;F.A.Lewis;R.L.Nantall.
  13. Science v.224 Evolution of proteolytic enzymes Neurath;H. https://doi.org/10.1126/science.6369538
  14. J. Korea Soc. Food Nutr. v.24 Purification and characterization of hemagglutinating protein from rhizome of alisma orientale Park;J.O.;K.S.Kim;K.-O. Sun Woo.
  15. J. L.,Reasearch monographs in cell and tissue physiology, elsenier science publishers B. V.(Biomedical Division) v.12 Inhibitors of serine proteinases. Power J.C.;J.W.Harper;Dingle J.T.(ed);Gordon(ed)
  16. Kor. J. Pharmacogn. v.27 Antimicrobial and antifungal studies on alismate rhizoma Toh;C.A.
  17. J. Biol. Chem. v.225 Inhibition of elastase and other serine proteases by heterocycling acylating agents Zimmerman;M;H.Morman;D.Mulvey;H.Jones;R.Frankshun;B.M.Aahe
  18. 현대의 한방Ⅰ v.167 현대 한방 연구소편 김정제