새롭게 합성한 크로모포릭 펩티드 기질에 의한 HIV - 1 프로테이자에의 효소반응속도 측정

윤주억,조경련 ( Joo Ok Yoon,Kyung Ryun Cho ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.4

HIV-1 protease hydrolyzed the newly synthesized peptides, Asn-Asn-Gln-Val-Phe (NO₂)-Val-Arg-NH₂ and acetyl-Arg-Lys-Leu-Val-Phe(NO₂)-Leu-Asp-Gly-NH₂ between the valyl and (p-nitro)phenylalanyl residues. The hydrolysis of these peptides resulted in a decrease in UV absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of these two peptide substrates was characterized by a linear time course at substrate turnover of ≤20%. The solubilities of these substrates at pH 4.7 were sufficient to perform initial rate measurements over a concentration range of 50 to 500 nM. Steady-state kinetic data and inhibition constants of the peptidolysis of these peptide substrates resulted in comparable values.

식품첨가물의 안전성에 관하여

윤주억 한국식품산업협회 1980 좋은식품 Vol.53 No.-

소의 Gastricsin 에 관한 연구

윤주억 東國大學校 1971 論文集 Vol.8-9 No.-

The proteolytic enzyme, gastricsin has been isolated from the mucosa of the fourth stomach (abomasum) of the cow. The gastricsin was purified by ammonium sulfate fractionation, chromatography on Amberlite CG-50, and gel filtration on Sephadex G-75. The preparation appeared to be homogeneous by zone electrophoresis and ultracentrifugation. The absorption spectrum of purified enzyme showed a maximum at 278mμ. the enzymic properties of bovine gastricsin, including optimal pH, proteolytic activity toward bovine hemoglobin, hydrolytic activities toward N-acetyl-L-phenylalanyl-L-phenylalanylglycine and N-acetyl-L-tyrosyl-L-tyrosylglycine as synthetic substrates, were distinguished from bovine pepsin. Bovine gastricsin did not hydrolyze the best synthetic substrate for pepsin, N-acetyl-L-phenylalanyl-L-phenylalanylgycine. Hydrolysis of N-acetyl-Ltyrosyl-L-tyrosylglycine by gastricsin occured at the L-tyrosyl-L-tyrosyl bond and was achieved without transpeptidation. With N-acetyl-phenylalany-L-phenylalanylglycine and N-acetyl-L-tyrosylglycine the solubilities in acid were much greater, and it was found possible to use a concentration of 12 mM without the adding of any organic solvent in the incubation mixture. The K_m of bovine gastricsin for N-acetyl-L-tyrosyl-L-tyrosylglycine was considerably higher and the K_cat considerably lower than the corresponding parameters of bovine pepsin for same substrate. Relative specificity of bovine gastricsin. k_cat/k_m for N-acetyl-L-tyrosyl-L-tyrosylglycine was considerably lower than bovine pepsin. The molecular weights for bovine gastricsin and pepsin were about 31,500 and 34,200, respectively, as determined by the sedimentation velocity method, and amino acid analysis data. The amino acid composition of bovine gastricsin differed significantly from those of bovine and porcine pepsin. Gastricsin contained a significantly higher number of glutamic acid residues than pepsin.

화경버섯에서 얻은 펩스타틴-불감응성 카르복시프로테이나아제의 기질 특이성

윤주억 동국대학교 산업기술연구원 1996 산업기술논문집 Vol.8 No.-

화경버섯(Lampteromyces japonicus)에서 처음으로 카르복시프로테이나아제(LCP)를 분리하였으며, 이 효소는 아스파르트산 프로테이나아제의 억제제인 펩스타틴과 diazoacetyl-DL-norleucine methylester에 대하여 감수성을 나타내지 않았다. LCP의 기질 특이성은 P5-P4-P3-P2-Phe-Nph-P2'-P3'(P5, P4, P3, P2, P2', P3'은 여러가지 아미노산 잔기를, Nph는 p-nitro-L-phenylalanine을 나타내며, 효소의 분해자라는 Phe-Nph 사이의 결합이다)와 같은 구조로 된 발색성 합성 펩티드 기질로서 연구하였다. 합성된 28가지 기질중, Lys-Val-Leu-Leu-Phe-Nph-Arg-Ala가 가장 최적 기질임이 밝혀졌다. 이 펩티드 기질에 대한 LCP의 효소반응 속도 상수들은 각각 K_m=15.5 μM, k_cat=12.4 s^-1, k_cat/K_m=0.80 μM^-1ㆍs^-1이었다. Tyrostatin은 LCP에 대하여 경쟁적 억제제 (Ki=2.1 nM)임이 밝혀졌다. Lampteromyces carboxyl proteinase (LCP), isolated from Lampteromyces japonicus, was the first example of a unique carboxyl proteinase which was obtained from mushrooms and was insensitive to aspartic proteinase inhibitors, such as pepstatin and diazoacetyl-DL-norleucine methylester. The substrate specificities of LCP was studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3'(P5, P4, P3, P2, P2', and P3' were a variety of amino acids, Nph was p-nitro-L-phenylalanine, and the Phe-Nph bond was cleaved). LCP was shown to hydrolyze a synthetic substrate, Lys-Val-Leu-Leu-Phe-Nph-Arg-Ala, most effectively among 28 substrates. The kinetic parameters of this peptide for LCP was K_m=15.5 μM, k_cat=12.4 s^-1, and k_cat / K_m=0.80 μM^-1ㆍs^-1ㆍ Tyrostatin was found to be a compeptitive inhibitor for LCP with a Ki value of 2.1 nM.

1,2-epoxy-3-(P-nitrophenoxy)propane에 의한 Gastricsin의 불활성화에 관한 연구

尹柱億 東國大學校 1977 論文集 Vol.16 No.-

소에서 단리한 gastricsin은 1, 2-epoxy -3-(p-nitrophenoxy) propance (EPNP)에 의하여 불활성화 되었고, 이 불성화된 효소 한 분자에는 EPNP 두 분자가 결합되었다. diazoacetyl-DL-norleucine methyl ester (DANM)나 EPNP로서 불활성화된 효소는, 활성효소와 같은 정도로, EPNP 또는 DANM과 각각 반응하였다. 또 효소분자에 결합된 DANM이나 EPNP는 히드록실아민 수용액으로 처리하면 그 전부 또는 일부를 제거할 수 있었다. 그러므로 gastricsin은 그 활성자리에 서로 다른 카르복시기 두개를 가지고 있으면서, 그중 하나는 DANM과, 또 하나는 EPNP와 반응하고, 이들 카르복시기는 다함께 효소반응에 관여하는 것으로 생각된다. Bovine gastricsin was inactivated by 1, 2-epoxy-3-(p-nitrophenoxy) propane (EPNP) with concomitant incorporation of two EPNP molecules per molecule of gastricsin. The diazoacetyl-DL-norleucine methyl ester (DANM)-inactivated enzyme and the EPNP-inactivated enzyme retained the same reactivities toward EPNP and DANM, respectively, as native gastricsin. The inactivators incorporated into the enzyme were labilized by treatment with aqueons hydroxylamine. Therefore, bovine gastricsin seems to have two distinct carboxyl groups in the active site, and they react with DANM and EPNP, respectively, and may participate cooperativly in the catalytic reaction.

Biosynthesis of Bovine Pepsinogen

윤주억,김성기,Yoon, Joo-Ok,Kim, Sung-Kih 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.3

소의 제4위 점막에서 전 RNA를 분리하고, 이 전 RNA로부터 oligo(dT)-cellulose 크로마토그라피와 Sepharose 4B 크로마로그라피로 poly(A) 함유 펩시노젠 mRNA를 정제하여 wheat germ cell-free system에서 펩시노젠 번역 반응을 실시하였다. 번역산물은 SDS-폴리아크릴아미드 겔 전기영동과 플루오로그라피로 분석한 결과, 주성분이 분자량 45,000과 43,000 돌턴의 두 가지 다른 분자형의 펩시노겐임이 밝혀졌다. In vitro 번역계에서 번역된 펩시노젠은 자촉척 활성화 기능이 없었다. 번역 펩시노겐의 카이오티립신과 Staphylococcus aureus V8 프로테아제 분해산물에 대한 peptide map은 소의 표준 펩시노젠에 대한 peptide map과 거의 일치하였다. Poly(A)-containing mRNA of pepsinogen was isolated from total RNA of bovine gastric mucosa by oligo (dT)-cellulose chromatography and Sepharose 4B chromatography, and was translated in a wheat germ cell-free system. The translation products. were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. A major translation product was pepsinogen which was synthesized in two different molecular forms with molecular weights of 45,000 and 43,000 daltons. The pepsinogen translated in the in vitro translation system had no autocatalytic activity. The peptidemaps of the translated pepsinogen which was digested by chymotrypsin and Staphylococcus aureus V8 protease were nearly identical with the corresponding peptide masps of standard bovine pepsinogen.

합성 Peptide Benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine 및 그 Ethyl Ester에 대한 pepsin작용

윤주억 동국대학교 농림과학연구소 1969 農林科學 論文集 Vol.3 No.-

The synthesis is described of new pepsin substrates of benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine and benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine ethyl ester for studies on the specificity of pepsin, and thin layer chromatographic examination of the peptides prepared showed that the new substrates are homogeneous. And also, same examination of the incubation mixtures showed that two synthetic substrates are cleaved by pepsin at the L-tyrosyl-L-phenylalanyl bond and hydrolysis of these substrates by pepsin is achieved without transpeptidation. It is found that synthetic peptides are moderately soluble with the amount of the substrate up to a concentration of 0.7 mM in aqueous sodium citrate buffers(0.04 M) in the pH range 1.8-4.0, thus obviating the necessity for the adding of an organic solvent in the assay mixture. The kinetic parameters for synthetic substrates are tabulated in the following table. The data in the table indicate that the susceptibility of synthetic peptides to peptic hydrolysis ◁표 삽입▷(원문을 참조하세요) are relatively large and the change of the carboxyl terminal group of synthetic substrate from glycine ethyl ester to glycine causes a small decrease in the susceptibility of the L-tyrosyl-L-phenylalanyl bond.

화경버섯의 항세균성 렉틴

윤주억,민태진,윤희식 ( Joo Ok Yoon,Tae Jin Min,Hee Sik Yoon ) 한국균학회 1995 韓國菌學會誌 Vol.23 No.1

소 팹시노젠의 생합성

윤주억,김성기 ( Joo Ok Yoon,Sung Kih Kim ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.3

Poly(A)-containing mRNA of pepsinogen was isolated from total RNA of bovine gastric mucosa by oligo (dT)-cellulose chromatography and Sepharose 4B chromatography, and was translated in a wheat germ cell-free system. The translation products. were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. A major translation product was pepsinogen which was synthesized in two different molecular forms with molecular weights of 45,000 and 43,000 daltons. The pepsinogen translated in the in vitro translation system had no autocatalytic activity. The peptidemaps of the translated pepsinogen which was digested by chymotrypsin and Staphylococcus aureus V8 protease were nearly identical with the corresponding peptide masps of standard bovine pepsinogen.

강아지 프로카이모신의 전 아미노산 서열 ( The Complete Amino Acid Sequence of Newborn Dog Prochymosin )

윤주억,김현구 동아시아식생활학회 1997 동아시아식생활학회지 Vol.7 No.3