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.

소의 펩시노오젠 cDNA 의 클로닝에 관한 연구

윤주억,김성기,정혜경 ( Joo Ok Yoon,Sung Ki Kim,Hye Kyoung Chung ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3

Bovine pepsinogen mRNA was partially purified from fourth stomach mucosa of cow. This preparation was used as the template for synthesis of cDNA on a pBR322-SV 40 hybrid plasmid DNA primer which was then circularized with linker DNA and used to transform E. coli. The resulting transformants were screened by in situ differential hybridzation using ^(32)P-labeled poly(A^+)RNA. Of 600 transformants screened, 8 were found to hybridize differentially; 1 of these contained an insert of about 900 base pairs and hybrid-selected a mRNA which directed synthesis of pepsinogen in a cell-free translation system. Using this cDNA as a probe, we studied the size of the major pepsinogen mRNA.

캘모듈린 cDNA 의 합성과 발현

윤주억,조경련,김성기 ( Joo Ok Yoon,Kyung Ryun Cho,Sung Kih Kim ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.3

A calmodulin cDNA was designed and synthesized from 61 chemically synthesized oligonucleotides using the method of enzymatic ligation and overproduced in Escherichia coli for the purpose of developing recombinant DNA approaches to study strucure-function relationships in this calcium-binding regulatory protein. The recombinant protein isolated from E. coli functions as a calmodulin in properties that were assayed: calcium binding and calcium-dependent conformational change. The initiating methionine was removed by E. coli leaving alanine as the first amino acid, as in the naturally occurring calmodulins. The first amino acid was not acetylated, but this difference from the higher plant and vertebrate calmodulins has no apparent effect on the function. The recombinant calmodulin also lacks posttranslational modification: a N^ε, N^ε, N^ε-trimethyllysine at position 115. The recombinant calmodulin was found to activate NAD kinase to a maximal level that was at least 3.8-fold higher than that obtained with soybean calmodulin. The lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.

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

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.

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.

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.

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.

좀개구리밥에서의 Histone 型 鹽基性 蛋白質의 單離

윤주억,신홍대,Yoon, Joo-Ok,Shin, Hong-Dae 대한화학회 1964 대한화학회지 Vol.8 No.4

"좀개구리밥"에서 histone 型 鹽基性 蛋白質을 單離하는데 처음으로 成功했다. 鹽基性 單白質은 "좀개구리밥"의 細胞磨碎物에서 稀 錦酸으로 抽出 分離하였다. 그 中 Amberlite CG-50에 (pH 6.0에서) 吸着되는 蛋白質成分은 송아지 胸腺 histone과 비슷한 amino 酸組成을 보였고, 構成 amino 酸 殘基로서는 lysine 이 特히 많았다. Carboxymethyl cellulose에 (pH 4.2에서) 吸着되는 蛋白成分의 溶出 chromatogram은 송아지 胸腺 histone과 비슷했으나, 各 peak의 比率은 相遠했으며, 特히 非吸着 成分이 含有되어 있음도 相違했다. 또 吸着成分의 amino 酸組成은 histone 型 鹽基成을 나타냈으나, 非吸着 成分은 典型的인 histone과는 相異한 蛋白質임을 確認했다. 송아지 胸腺 histone과 "좀개구리밥"에서 單離한 蛋白 成分들의 黃酸 存在下에서 $HgSO_4$와의 沈澱反應을 histone아닌 蛋白質과 比較 檢討한 結果, "좀개구리밥"에서 單離한 것은 histone 型 蛋白成分이 分明하였다. We isolated histone-type basic proteins from lemna paucicostata for the first time. Basic proteins were extracted directly with dilute mineral acids from homogenized lemna paucicostata. Amino acid compositions of basic protein portions adsorbed on Amberlite CG-50(at pH 6. 0) were resembled to those of calf thymus histones. Especially, lysine content was the greatest of the other amino acids. By chromatographic studies, adsorbed portions of basic protein components on carboxymethyl cellulose column(at pH 4. 2) were shown to be homogeneous to calf thymus histones, however, the area under the individual curve was different, and furthermore, the containing of a non-adsorbed portion in the large extent was markedly different from calf thymus histones. And amino acid compositions of adsorbed portions represented the histone-type basic propertes, but non-adsorbed portions were considered as a different protein compared with the typical histone. When calf thymus histone and protein components separated from lemna paucicostata were heated($60^{\circ}C$) with a solution of $HgSO_4-H_2SO_4$, precipitates were not obtained.

콩 캘모듈린의 아미노산 서열

윤주억,조경련,변광의 ( Joo Ok Yoon,Kyung Ryun Cho,Kwang Eui Byoun ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

The complete amino acid sequence of calmodulin from soybean was determined by purifying and sequencing the cyanogen bromide and tryptic peptides. Soybean calmodulin consisted of 148 amino acid residues and its aminoterminus was blocked with an acetyl group. This calmodulin lacked tryptophan and contained one mol each of N ?trimethyllysine, histidine, and cysteine residues and two moles of tyrosine residues per mol of the protein. The comparison of the amino acid sequence of soybean calmodulin with that of bovine brain calmodulin indicated that there were nine amino acid substitutions other than amide assignments, one insertion and one deletion of amino acid residues in soybean calmodulin.