Competitive Inhibition of Pepsin by Carboxylic Acids

신홍대,Hong Dae Shin Korean Chemical Society 1970 대한화학회지 Vol.14 No.2

In order to obtain the more effective evidence, supporting the hypothesis which have been previously described by former report that pepsin (EC 3.4. 4.1) forms a hydrophobic bond with the nonpolar side chain of its substrate, the inhibitory effect of carboxylic acids(from formic acid to iso-butyric acid) on the activity of pepsin to the synthetic dipeptide, N-Carbobenzoxy-L-glutamyl-L-tyrosine, was discussed. The kinetic study showed that the inhibition by carboxylic acids was competitive. The Kidecreased with increasing size of the inhibitor molecule. The $-{\Delta}F^{\circ}$increased linearly with increasing number of carbon atoms in the hydrocarbon chain of the inhibitor. It was confirmed that the hydrophobic bond between more than one side chain of amino acid residues(phenylalanine) in the binding region of the active center of pepsin and the side chain of amino acid residues in the substrate was formed as the first step of its enzymic mechanism. The inhibitory effect of carboxylic acids was due to the competition of the hydrocarbon group of the carboxylic acids with the side chain of the substrate for the hydrophobic binding site(the side chain of phenylalanine) of the pepsin.

The Kinetics of the Pepsin-Catalyzed Hydrolysis of N-Carbobenzoxy-L-Glutamyl-L-Tyrosine by Determination of the Spectrophotometer

신홍대,Hong Dae Shin Korean Chemical Society 1970 대한화학회지 Vol.14 No.2

The kinetics of the pepsin-catalyzed hydrolysis of N-carbobenzoxy-L-glutamyl-L-tyrosine at pH 3.5 and $37^{\circ}C$ were determined by a spectrophotometric technique. The pepsin used was further purified on a Sephadex G-75 column. The kinetics data were Km = l.7 ${\times}10^{-3}M,\;-{\Delta}F^{\circ}$ = 3.99Kcal/mole, and $k^3=\;2.1{\times}10^{-2}\;sec^{-1}$. An analysis of the above data and other investigators' data obtained from some dipeptides led to the following conclusions. (1) Phenylalanyl residues in a synthetic peptide are bound to pepsin more strongly than glutamyl or tyrosyl residues, supporting the theory that a part of the binding region of the active center is hydrophobic. (2) Dipeptides are bound to pepsin principally through their side chains and the binding involves both side-chain residues. (3) The nature of amino acids in dipeptides $R_2-R_1,\;affect\;the\;k_3$ values.

Sodium Alginate-$CaSO_4{\cdot}1/2H_2O$ 彈性體의 生成速度 및 彈性에 미치는 金屬酸化物의 影響

신홍대,허연,김은식,Shin, Hong-Dae,Huh, Yeonn,Kim, Uun-Sik 대한화학회 1965 대한화학회지 Vol.9 No.1

Sodium alginate-$CaSO_4{\cdot}$1/2 $H_2O$의 gel에 미치는 alkali鹽의 遲延효果를 Miyake date와 比較하고, Schwedoff法을 改良한 連續的 測定方法을 써서, 金屬酸化物의 添加에 依한 彈性 gel의 生成速度 및 彈性의 變化를 硏究하였다. 즉 (1) sample의 張力變化를 連續的으로 測定하여, sodium alginate와 $CaSO_4{\cdot}$1/2 $H_2O$ aqueous sol의 gel化 過程을 觀察하였다. (2) 彈性 gel의 生成速度에 미치는 $Na_3PO_4$의 遲延효果는 $Na_2CO_3$보다 크다는 點에서는 Miyake 報告와 一致하였으나, Miyake date처럼 兩者間에 그리 큰 差가 없었다. (3) 金屬酸化物이 彈性 gel生成速度에 미치는 影響에 어떤 規則性은 없었지마는 $SiO_2$와 MgO는 比較的 큰 速度增大효果를 나타내고 ZnO는 比較的 적었다. 그러나 $Al_2O_3$, $Sb_2O_3$와 $TiO_2$는 速度低下효果를 나타냈다. 結果的으로 酸化物의 種類나 그 添加量에 따라 어떤 明確한 規則性을 觀察하지 못하였다. (4) 彈性率과 生成速度에 미치는 효果사이에 比例關係는 없없으나 金屬酸化物을 添加하면 一般的으로 彈性率이 增加하였다. 이들 酸化物 中에서 彈性率增大효果가 가장 큰 것은 MgO였다. The time-lag-effect of alkali salts on the gelation of sodium alginate-$CaSO_4{\cdot}$1/2 $H_2O$ is compared with Miyake's data, and then the formation rate of the elastics measured by the continuous method (an improved Schwedoff's method) and the change of rigidity with metallic oxides are studied as follows: (1) The gelation processes of sodium alginate and $CaSO_4{\cdot}$1/2 $H_2O$-aqueous sol are studied by measuring, continuously the increases of tensions ofsamples. (2) The time-lag-effect of $Na_3PO_4$ on the formation rate of the elastic gel is larger than that of $Na_2CO_3$, but the difference between the effects of the two alkali salts on the rate is found not so greater than predicted in Miyake's data. (3) Any regularities of the effect on the rate by metallic oxides are not observed. The increasing effects of the rates of $SiO_2$ and MgO are relatively large, and that of ZnO is relatively small. However, $Al_2O_3$, $Sb_2O_3$ and $TiO_2$show some decreasing effects. As a result it is noted that the regularities do not depend on the effect of oxide species and their amounts. (4) It is not found proportionality between the rigidity and the gelation rate. However, the increasing effect of the rigidity with the addition of metallic oxides can be observed. The rigidity increasing rate of MgO is the largest of them.

막장의 아미노酸 組成에 關한 硏究

신홍대,윤주억,Hong Dae Shin,Joo Ok Yoon 대한화학회 1963 대한화학회지 Vol.7 No.1

Amino acid composition of Makjang was determined by combined usage of ion exchanged resin and paper chormatography in the following states. a. A fraction soluble in water b. Hydrolysate of the whole Makjang c. Same as a. (p.p.t. formed by tungstic acid or trichloroacetic acid being removed) d. Hydrolysate of c. (T and TCA) By comparing amino acid composition of Makjang with that of its raw material, we found that decomposition of essential amino acids during brewing is slight. From the amino acid composition of a,b,c,d, we discussed the ratio of amino acid liberation during brewing and assumed that Makjang contains peptide-like substances composing of glutamic acid and aspartic acid.

Mannich Reaction을 利用한 Bis-(-β-benzoylethyl) aniline hydrochloride의 preparation에 關한 硏究

申洪大(Hong Dae Shin) 부산대학교 인문학연구소 1962 코기토 Vol.5 No.-

새로운 합성 펩티드에 대한 펩신 작용 1. Benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine 과 그의 에틸에스테르에 대한 펩신 작용

윤주억,신홍대,Yoon, Joo-Ok,Shin, Hong-Dae 대한화학회 1969 대한화학회지 Vol.13 No.3

The synthesis is described of new pepsin substrates of benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine ethyl ester and benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine for studies on the specificity of pepsin, and thin layer chromatographic examination of the peptides prepared showed 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 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.

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.

Vibrio Vulnificus Lipopolysaccharide의 생물활성과 그에 미치는 Cell Wall Protein-A의 영향

김용호,신홍대,강신원,Kim, Yong-Ho,Shin, Hong-Dae,Kang, Shin-Won 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3

Vibrio(V.) vulnificus 대량 배양에 의하여 회수된 균체로부터 lipopolysaccharide(LPS)와 cell wall protein-A(CWP-A)를 추출하여 Sprague Dawley rat에서 생물활성을 생체의 독소 배설 기능에 관련된 장기의 효소 및 혈중 성분을 비교 측정한 결과 aspartate transaminase(AST), alanine aminotransferase(ALT) 및 blood urea nitrogen(BUN)은 LPS쪽의 활성이 높았으나 lactic dehydrogenase(LDH)는 CWP-A쪽이 훨씬 높았다. 기타의 측정군은 양자에서 모두 유사한 활성을 나타내었다. 따라서 숙주 혈중 순환 효소 및 성분의 변동 성향을 고려해 볼 때 V. vulnificus 병독성 해석에 내독소인 LPS 뿐만 아니라 CWP-A도 중요하게 고려되어야 한다고 본다. Lipopolysaccaride(LPS) and cell wall protein-A(CWP-A) were extracted from cell wall of Vibrio(V.) vulnificus. The biological activities of CWP-A were weaker than LPS in aspartate transaminase(ASP), alanine aminotransferase(ALT) and blood urea nitrogen(BUN), but more strong in lactic dehydrogenase(LDH) and very similiar in other test. CWP-A of V. vulnificus were considered to be important for the expression of virulence, LPS as well.

Vibrio Vulnificus Lipopolysaccharide 의 생물활성과 그에 미치는 Cell Wall Protein - A 의 영향

김용호,신홍대,강신원 ( Yong Ho Kim,Hong Dae Shin,Shin Won Kang ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3

Lipopolysaccaride(LPS) and cell wall protein-A(CWP-A) were extracted from cell wall of Vibrio(V.) vulnificus. The biological activities of CWP-A were weaker than LPS in aspartate transaminase(ASP), alanine aminotransferase(ALT) and blood urea nitrogen(BUN), but more strong in lactic dehydrogenase (LDH) and very similiar in other test. CWP-A of V. vulnificus were considered to be important for the expression of virulence, LPS as well.

Cyclic Nona 및 Pentapeptide 유도체들의 합성과 단백질 분해 효소에 대한 저해활성 측정

강신원,허남원,신홍대 ( Shin Won Kang,Nam Won Huh,Hong Dae Shin ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.4

Most of legume seeds contain serveral kinds of BBI-type inhibitors. They have similar amino acid residues around the reactive site. The P₁ site amino acid residue in reactive site generally corresponds to the specificity of the cognate enzyme. It has also been noted the importance of P₁ site amino acid residue in synthetic cyclic nonapeptide derivatives which were related to active site of BBI. To obtain further understanding of the role of amino acid residues near th reactive site, 3 cyclic nonapeptides which are substituted analogues of GBI-II were synthesized and the inhibition constants for some proteinases were calculated by Dixon method. Antielastic fragment in GBI-II (I_a) differ only P₂ Site with that of soybean isoinhibitor C-II, Ki of Ia for porcine pancreatic elastase was 48μM, Ki of antitryptic fragment in LBI (I_b) for trypsin was 3.7μM, and Tyr analogue (I_c) for chymotrypsin showed no inhibition but appeared low inhibitory activity for trypsin and elastase. To elucidate the relation between loop size and inhibitory activity, 3 cyclic pentapeptide analogues containing the each reactive site of Ala-Ser, Lys-Ser and Tyr-Ser were synthesized. These peptides possessed slight inhibitory activity toward proteinases, Ki of synthetic cyclic pentapeptides for elastase, trypsin, and chymotrypsin were 614μM, 278μM and 128μM, respectively.