Dihydropyridine Dinucleotide ( NADH ) 와 탈수소 효소의 결합 반응에 관한 연구

김수자,이현재 ( Soo Ja Kim,Hyun Jae Lee ) 생화학분자생물학회 1976 BMB Reports Vol.9 No.1

The L-α-glycerophosphate dehydrogenase-catalyzed reduction of dihydroxyacetone phosphate was studied to investigate the properties of NADHdehydrogenase complex. A series of oxidized coenzyme (NAD) competitive inhibitors were employed for the study of inhibition on the reverse reaction of NAD-linked dehydrogenase systems. Inhibition by adenosine derivatives and carboxylic acids was competitive with respect to NADH, and by N¹-alkylnicotinamide chlorides and n-alkyl-ammonium chlorides was non-competitive with respect to the reduced coenzyme. The dihydronicotinamide ring of NADH was demonstrated not to interact with the pyridinium ring region, but was proposed to interact with the hydrophobic region instead.

단백질의 화학적 변형

김수자,이현재 ( Soo Ja Kim,Hyun Jae Lee ) 한국생화학분자생물학회 (구 한국생화학회) 1986 생화학총설집 Vol.1 No.-

Inactivation Study of Pyridine-Linked Dehydrogenases by $N^1$-Alkylnicotinamide Chlorides

김수자,이현재,Kim Soo-Ja,Lee Hyun Jae Korean Chemical Society 1976 대한화학회지 Vol.20 No.5

A series of $N^1$-alkylnicotinamide chlorides, $N^1$-methyl-to $N^1$-dodecylnicotinamides inclusive were studied with rabbit muscle L-${\alpha}$-glycerophosphate dehydrogenase to investigate the possibility of reversible and irreversible inactivation of the pyridine-linked dehydrogenases by the coenzyme-competitive inhibitor derivatives. The inhibition of the enzyme by $N^1$-alkylnicotinamide chlorides was demonstrated to be reversible at the dilute concentration of the inhibitors but this reversible inhibition was found to be followed by an irreversible time-dependent inactivation measuable at high concentrations of the inhibitors. The properties of this time-dependent inactivation were discussed on the basis of the denaturation of the enzyme by the binding of small micelle-like structures formed at higher concentrations of the inhibitors. Pyridine관여 탈수소 효소는 $N^1$-alkylnicotinamide chloride 유도체에 의하여 저해 작용을 받고 있는 바 저해제이 농도 변화에 따른 효소 저해작용이 가역 또는 비가역 불활성화 반응에 기인하는지의 여부를 밝혀 보기 위하여 토끼 근육으로 부터 유리한 L-${\alpha}$-glycerophosphate dehydrogenase를 사용하여 연구하였다. 이 효소의 저해작용은 상용한 저해제 유도체의 농도가 희박했을 경우 가역적인 효소저해 반응을 보여주고 있으나 저해제의 농도가 증가함에 따라 점차 비가역적인 효소 불활성화로서 나타남을 알았으며 이러한 비가역 불활성화 반응은 저해제의 농도가 증가함에 따라 형성될 수 있는 micelle 구조의 미세분자와의 결합에 의한 효소의 변성에 기인할 것이라고 결론을 얻었다.

Plasminogen 활성제로서의 Urokinase 의 활성화 부위내 작용기들에 대한 연구

박진우,김수자,이현재 ( Jin Woo Park,Soo Ja Kim,Hyun Jae Lee ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.2

In order to elucidate the mechanism of the urokinase- induced plasminogen activetion, catalytically active functional groups which involved in the active site of urokinase molecule were investigated by a series of kinetic and chemical modification experiments. Inhibition study with several lysine and arginine derivatives indicates there involves hydrophobic interaction between urokinase and plasminogen molecules. 6-Amino hexanoic acid shows an exceptional high degree inhibition. The results from a set of pH-activity profiles and several chamical modification experiments suggest there involve at lease one serine hydroxyl moiety and one histidine imidazole group in the active site of the activator urokinase for the catalytic conversion of plaminogen into plasmin. Based on the results obtained, a minimal three step mechanism of the UK-induced plasminogen activation process was proposed as a working model.

Studies on the Plasminogen Activating Enzymes: I. A Comparative Study on the Activation of Human Plasminogen by Humoral and Bacterial Activators

민흥식,김수자,이현재,Min, Heung-Sik,Kim, Soo-Ja,Lee, Hyun-Jae 생화학분자생물학회 1980 한국생화학회지 Vol.13 No.4

프라스미노겐 활성화 반응을 촉진하는 미생물 활성제로서 Streptokinase (SK)와 Staphylokinase (SLK)를 새로히 선별하여 얻은 두 종의 병원성박테리아인 Streptococci sp.와 Staphylococci S104로부터 각각 분리. 정제하였으며, 이들 박테리아 활성제들의 반응 특이성을 사람의 오줌으로부터 얻은 Urokinase (UK)의 것과 비교 연구하였다. 박테리아 활성제인 SK와 SLK는 서로 비슷한 양상의 특성을 띄우며, 단백질 분해 효소로서의 촉매기능에 있어 프라스미노겐만을 기질로 요구한다는 점에 있어서는 UK와 비슷하나 에스테르 가수분해 기능에 있어서는 상이함을 알 수 있었다. 특히, 이들 박테리아 활성제는 기질인 프라스미노겐 농도에 따른 반응속도의 변화 양상에 있어 UK에서와는 상이하게 만곡형의 증가를 보여주고 있으며 또한 이들에 의한 프라스미노겐 활성화 반응이 Casein이나 알부민(BSA) 첨가에 의하여 저해를 받고 있다는 점에서도 UK와는 구별될 수 있었다. 이러한 결과를 토대로 SK와 SLK 등의 박테리아 활성제는 프라스미노겐과의 결합반응을 위해 두 개의 결합 부위가 있는 것으로 추측되며 이중 한 부위에서의 기질결합 중간체는 프라스미노겐 활성화를 보다 촉진시키는 것으로 보았다. A comparison was made of the proteolytic and esteratic potencies of bacterial activators and urokinase, and the results along with some kinetic and inhibition study suggest that the modes of the plasminogen activation by bacterial activators are different from that of urokinase. Two forms of bacterial activator, stretokinase(SK) and staphylokinase (SLK), were isolated from the newly screened $\beta$-hemolytic pathogenic bacteria of Streptococci sp. and Staphylococci S104, respectively, and were purified to give homogeneous preparations. Both forms of bacterial activator showed considerable degrees of the proteolytic activities, but their substrate specifities were found to be very limited toward plasminogen as in the case of urokinase. However, unlike urokinase, bacterial activators did not exhibited any esterase activity at all. The Km values for bacterial activators and urokinase were estimated to be about $1{\times}10^{-7}M$ and $2.2{\times}10^{-6}M$, respectively, with human plasminogen as a substrate. The kinetic rate profiles against substrate concentration showed sigmoid curves for SK and SLK while a hyperolic saturation curve for urokinase. Furthermore, the proteolytic or plasminogenic activites of SK and SLK were demonstrated to be inhibited by macromolecular proteins such as casein and bovine serum albumin. Based on the results obtained, it was suggested that bacterial activators may have two substrate binding sites and thus one of them may serve to provide the more active species by forming an intermediate complex with the first molecule of plasminogen.

Active Site Functional Residues of Urokinase as a Plasminogen Activator

박진우,김수자,이현재,Park, Jin-Woo,Kim, Soc-Ja,Lee, Hyun-Jae 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.2

In order to elucidate the mechanism of the urokinase- induced plasminogen activetion, catalytically active functional groups which involved in the active site of urokinase molecule were investigated by a series of kinetic and chemical modification experiments. Inhibition study with several lysine and arginine derivatives indicates there involves hydrophobic interaction between urokinase and plasminogen molecules. 6-Amino hexanoic acid shows an exceptional high degree inhibition. The results from a set of pH-activity profiles and several chamical modification experiments suggest there involve at lease one serine hydroxyl moiety and one histidine imidazole group in the active site of the activator urokinase for the catalytic conversion of plaminogen into plasmin. Based on the results obtained, a minimal three step mechanism of the UK-induced plasminogen activation process was proposed as a working model. Urokinase에 의한 plasminogen 활성화 반응은 urokinase-plasminogen 복합체인 acyl-중간체를 경유하는 것으로 믿어지며, urokinase 분자내 활성화 부위 작용기로서는 histidine imidazole 잔기와 serine hydroxyl가 각각 염기성 및 천핵성 촉매제로서 작용, plasminogen의 활성화 반응에 참여될 수 있음을 pH 변화에 따른 반응 속도론적 실험과 착용기에 대한 화학적 불활성화 실험을 통하여 입증할 수 있었다. 아울러 본 연구결과를 토대로 하여 urokinase에 의하여 유도되는 plasminogen 활성화 반응에 대한 3단계 반응 메카니즘도 구상 검토해 보았다.

프라스마겐 활성화 효소에 관한 연구 Ⅰ. 박테리아 및 체액의 활성제에 의한 프라스미노겐 활성화 반응의 비교연구

민흥식,김수자,이현재 ( Heung Sik Min,Soo Ja Kim,Hyun Jae Lee ) 생화학분자생물학회 1980 BMB Reports Vol.13 No.4

A comparison was made of the proteolytic and esteratic potencies of bacterial activators and urokinase, and the results along with some kinetic and inhibition study suggest that the modes of the plasminogen activation by bacterial activators are different from that of urokinase. Two forms of bacterial activator, stretokinase(SK) and ataphylokinase (SLK), were isolated from the newly screened β-hemolytic pathogenic bacteria of Streptococci sp. and Staphylococci S104, respectively, and were purified to give homogeneous preparations. Both forms of bacterial activator showed considerable degrees of the proteolytic activities, but their substrate specifities were found to be very limited toward plasminogen as in the case of urokinase. However, unlike urokinase, bacterial activators did not exhibited any esterase activity at all. The Km values for bacterial activators and urokinase were estimated to be about 1×10^(-7)TM and 2.2×10^(-6)M, respectively, with human plasminogen as a substrate. The kinetic rate profiles against substrate concentration showed sigmoid curves for SK and SLK while a hyperolic saturation curve for urokinase. Furthermore, the proteolytic or plasminogenic activites of SK and SLK were demonstrated to be inhibited by macromolecular proteins such as casein and bovine serum albumin. Based on the results obtained, it was suggested that bacterial activators may have two substrate binding sites and thus one of them may serve to provide the more active species by forming an intermediate complex with the first molecule of plasminogen.

광 박테리아 색소체의 고정화에 의한 인위적 광 화학 반응조의 제조 및 응용

이현재,이동희,김수자 ( Hyun Jae Lee,Dong Hee Lee,Soo Ja Kim ) 생화학분자생물학회 1981 BMB Reports Vol.14 No.3

The bacterial chromatophores prepared from R. rubrum was immobilized by polyacrylamide entrapment to provide an efficient photosynthetic reactor for the generation of ATP and the reducing powers. The optimum yield of the immobilized chromatophores was obtained at the monomer concentration of 7.5% with its one-tenth amount of the crossfinking agent, The stability of chromatophores was improved markedly by the immobilization. The immobilized chromatophores showed the broad pH and temperature optimum at the pH range of 6.0 to 8.0 and at 40∼45℃, respectively, The temperature dependent deactivation constants for the native and immobilized chromatophores were estimated to be about 3.5×10-2 and 8.7×10-` per min, respectively, at 40℃. By immobilization, the original chromatophore activities including photophosphorylation of ADP and photoreduction of NAD^+ were increased markedly and the operational stability was also improved. The Km values of the photophosphorylation by the immobilized chromatophores were estimated to be 4.6×10^(-4) M and 2.7×10^(-3) M for ADP and inorganic phosphate, respectively, which were about 15 to 30 times higher than those for the native chromatophores, In addition, in the case of the succinatelinked photoreduction of NAD^+, the immobilization of chromatophores resulted in a marked stimulation, especially by the presence of KCN and oligomycin. Several other properties related to the photochemical reactions mediated by the immobilized chromatophores were also studied and compared with those of the native chromatophores.

Preparation and Application of Photosynthetic Reactor : Immobilization of Bacterial Chromatophores

이현재,이동희,김수자,Lee, Hyun-Jae,Lee, Dong-Hee,Kim, Soo-Ja 생화학분자생물학회 1981 한국생화학회지 Vol.14 No.3

The bacterial chromatophores prepared from R. rubrum was immobilized by polyacrylamide entrapment to provide an efficient photosynthetic reactor for the generation of ATP and the reducing powers. The optimum yield of the immobilized chromatophores was obtained at the monomer concentration of 7.5% with its one-tenth amount of the crossfinking agent, The stability of chromatophores was improved markedly by the immobilization. The immobilized chromatophores showed the broad pH and temperature optimum at the pH range of 6.0 to 8.0 and at $40{\sim}45^{\circ}C$, respectively, The temperature dependent deactivation constants for the native and immobilized chromatophores were estimated to be about $3.5{\times}10^{-2}$ and $8.7{\times}10^{-3}$ per min, respectively, at $40^{\circ}C$. By immobilization, the original chromatophore activities including photophosphorylation of ADP and photoreduction of $NAD^+$ were increased markedly and the operational stability was also improved. The $K_m$ values of the photophosphorylation by the immobilized chromatophores were estimated to be $4.6{\times}10^{-4}M$ and $2.7{\times}10^{-3}M$ for ADP and inorganic phosphate, respectively, which were about 15 to 30 times higher than those for the native chromatophores, In addition, in the case of the succinatelinked photoreduction of $NAD^+$, the immobilization of chromatophores resulted in a marked stimulation, especially by the presence of KCN and oligomycin. Several other properties related to the photochemical reactions mediated by the immobilized chromatophores were also studied and compared with those of the native chromatophores. 광합성 박테리아인 Rhodospirillum rubrum의 색소체를 폴리아크릴아미드에 흡입시켜 고정화 반응을 일으켰으며, 이 경우 색소체 고정화의 최대수율은 아크릴아미드 단당체의 농도가 7.5%이고 교차 결합도가 약 10%일 경우에 얻어졌다. 광 박테리아 색소체가 지닌 광 인산화 반응과 광 환원 반응의 활성도는 색소체의 고정화 반응으로 상당한 증가를 보였을 뿐 아니라 고정화 반응에 의하여 색소체의 안정도가 크게 증진되었음을 알았다. 즉 색소체의 고정화에 의하여 광 반응의 최적 pH 및 최적온도의 범위가 확대되었으며 온도에 대한 색소체의 불활성화상수 $(K_d)$ 값은 유리상태와 고정화된 색소체의 경우 각각 $3.5{\times}10^{-2}$/min과 $8.7{\times}10^{-3}$/min로 얻어졌다. 광 인산화 반응의 $K_m$ 값은 색소체의 고정화로 인하여 약 15~30배 정도 증가하였으며 고정화 색소체에 의한 값은 ADP와 무기인산에 대하여 각각 $4.6{\times}10^{-4}M$과 $2.7{\times}10^{-3}M$로 얻어졌다. 한편 광 환원 반응에 있어서도 색소체의 고정화에 의하여 활성도가 증진되었으며 특히 KCN과 oligomycin 존재 하에서 커다란 증가차이를 보여 주었다. 그밖의 유리상태와 고정화된 색소체가 지닌 광 화학 반응의 특성을 비교 검토하였으며 고정화원 색소체의 응용 가능성을 논하였다.

Studies on the Anticoagulation Process (Fibrinolysis) by Urokinase I. Purification and Characterization of Urokinase from Human Urine

박진우,이현재,김수자,Park, Jin-Woo,Lee, Hyun-Jae,Kim, Soo-Ja 생화학분자생물학회 1980 한국생화학회지 Vol.13 No.3

Urokinase는 비활성 혈장단백질인 프라스민 전구체를 활성화시키는 효소로서, 이 효소반응의 산물인 프라스민이 혈류의 응혈요소인 피브린 가수분해 과정에 참여될 수 있음으로 이 효소, 즉 Urokinase가 지닌 특성을 연주 검토하였다. 효소 시료의 정제는 ECTEOLA 수지에 의한 이온 교환 크로마토그래피 방법과 알지닌 또는 아그마틴을 ligand로 하는 친화성 크로마토그래피 방법을 도입하여 약 60배의 정제도를 얻었으며 이 경우 효소의 비활성도는 약 1,300 units/mg protein 이였다. 효소의 특성 중 특기할만한 사실은 기질 특성으로서, 다른 단백질 분해 효소인 trypsin과 작용은 비슷하나 프라스민 전구체인 프라스민노젠에만 특이하게 작용함을 알았으며 효소활성도의 최적 pH와 최적온도 등에 있어서도 다른 유사단백질 분해효소들과 다름을 알았다. 그 밖의 효소 특성인 기질에 대한 친화력 및 저해제에 의한 결과 등을 토대로 이 효소가 지난 생리적 역할, 즉 피브린 가수분해 조절기능에 대하여도 검토하여 보았다. The activation of plasminogen to an active fibrinolytic enzyme, plasmin, was studied withe urokinase(EC 3.4.26.99). A crude form of urokinase obtained from human urine was purified partially by an ion exchange column of ECTEOLA and further by an affinity column employing arginine or agmatine as a ligand on Sepharose 4B matrix. The specific activity of the final enzyme preparation was about 1,300 units per ㎎ protein based on the caseinolytic assay measuring the rate of casein hydrolysis by plasmin formed. The mode of the enzyme action seems to be similar to that of trypsin in term of the esterase activity, but the enzyme is found to be very specific toward plasminogen as the substrate. Characteristic properties of the enzyme including molecular weight, pH and temderature optimum, and some kinetic properties were studied and compared with that of the other proteolytic enzyme of trypsin and plasmin. In addition, based on its associative properties, the nature and the physiological role in the fibrinolysis were also discussed.