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.

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.

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.

소의 Pepsinogen의 생합성

윤주억,김성기,Yoon, Joo-Ok,Kim, Sung-Kin 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.1

소의 제4위점막의 total RNA로부터 oligo(dT)-cellulose 크로마토그라피와 Sepharose 4B 크로마토그라피로 poly(A) 함유 pepsinogen mRNA를 분리 정제하고, wheat germ cell-free system에서 pepsinogen translation반응을 실시하였다. Translation product는 sodium dodecyl sulfate-polyacrylamide gel 전기영동과 fluorgraphy로 분석한 결과 주성분이 분자량 45,000과 43,000 dalton의 두가지 다른 분자형의 pepsinogen임이 밝혀졌다. 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 $^4$sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. A major translation product was pepsinogen which was synthesized as two different molecular forms with molecular weights of 45,000 and 43,000 daltons.

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.

Molecular Cloning of a cDNA Sequence for Bovine Pepsinogen

윤주억,김성기,정혜경,Yoon, Joo-Ok,Kim, Sung-Ki,Chung, Hye-Kyoung 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3

소의 제4위점막으로부터 펩시노오젠 mRNA를 부분정제 하였다. 이것은 pBR322-SV 40 하이브리드 플라스마드 DNA 프라이머 위에 소의 펩시노오젠 cDNA을 합성할 때 주형으로 삼았으며, 링커 DNA로서 고리화하고 E. coli (DH 1)의 형질전환에 사용하였다. 얻어진 형질전환 세포는 $^{32}P$-표지 poly($A^+$)RNA를 사용하여 in situ differential 하이브리드 형성반응으로 스크린하였다. 스크린된 600의 형질전환 세 포 가운데, 8 클론이 differential 양성으로 나타났고, 그 중 하나가 약 900 염기쌍으로 된 insert를 가지고 있었으며, 하이브리드-선택 번역반응으로 펩시노오젠을 합성하는 mRNA 임이 밝혀졌다. 이 cDNA를 probe로 사용하여 소의 주요 펩시노오젠 mRNA의 크기를 알아보았다. 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.

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.

오이 모자이크 바이러스 피복 단백질의 아미노-말단 구조

윤주억,Yoon, Joo-Ok 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.1

오이 모자이크 바이러스 B형 피복 단백질의 아마노-말단 구조를, 5 nmol의 아미노-말단 랩티투로서 간단한 수동식 Edman 분해로 결정하였다. 이 방법은 신속하고 정량적으로 anilinothiazolinone을 얻고 15분간 역가수분해하여 아미노산 분석을 하거나, phenylthiohydantoin 아미노산으로 전환시킨 후 고속 액체크로마토그라피로 분석하는 것 이다. 피복 단백질의 아미노-말단 잔기는 아세틸화되어 있었으므로, 효소분해와 역상계 고속 액체 크로마토그그라피를 병용하여 동정하였다. 피복 단백질의 아미노-말단 구조(14잔기 까지)는 다음과 같다. Acetyl-Met-Asp-Asp-His-Glu-Leu-Thr-Leu-Ser-Tyr-Ala-Ala-Ser-Lys-. The amino-terminal sequence of the B-type coat protein from cucumber mosaic virus was determined by a simple manual Edman degradation with 5 nmol of amino-terminal peptide. This was achieved by a fast, quantitative method of obtaining the anilinothiazolinone or phenylthiohydantoin amion acid, and quantitations were made by either a rapid 15 min-back hydrolysis and amino acid analysis or by a high-performance liquid chromatographic procedure. The amino-terminal residue of the coat protein was acetylated and was identified by a combination of enzymatic cleavage and reversephase high-performance liquid chromatography. On the basis of the analytical data, the amino-terminal sequence of 14 residues was established to be acetyl-Met-Asp-Asp-His-Glu-Leu-Thr-Leu-Ser-Tyr-Ala-Ala-Ser-Lys-.

HIV-1 Protease Assay by the Newly Synthesized Chromophoric Peptide Substrates

윤주억,조경련,Yoon, Joo-Ok,Cho, Kyung-Ryun Korean Society for Biochemistry and Molecular Biol 1991 한국생화학회지 Vol.24 No.4

HIV-1 프로테아제의 새로운 크로모포릭 펩티드 기질로서 Asn-Asn-Gln-Val-Phe$(NO_2)$-ValArg-$NH_2$와 acetyl-Arg-Lys-Leu-Val-Phe$(NO_2)$-Leu-Asp-Gly-$NH_2$를 합성하고, 발린과 p-니트로 페닐알라닌 잔기 사이의 펩티드 결합이 가수분해됨을 알았다. 이들 합성 펩티드는 분해되면 310 nm에서의 흡광도가 감소되었다. HIV-1 프로테아제의 두 펩티드 기질의 가수분해 반응속도는 기질 턴오버수 ${\leq}$20%에서 반응시간에 비례하였다. 이들 합성기질의 용해도는 pH 4.7, 50-500 nM에서 반응 초속도 측정에 충분하였다. 또 합성한 두 펩티드 기질에 대한 정상상태에서의 효소반응이 $K_{cat}$, $K_m$, $K_{cat}/K_m$ 및 $K_i$값들을 다른 펩티드 기질에 대한 값들과 비교 검토하였다. HIV-1 protease hydrolyzed the newly synthesized peptides, Asn-Asn-Gln-Val-Phe $(NO_2)$-Val-Arg-$NH_2$ and acetyl-Arg-Lys-Leu-Val-Phe$(NO_2)$-Leu-Asp-Gly-$NH_2$ between the valyl and $({\rho}-nitro)$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 ${\leq}$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.