단세포 녹조류 Chlamydomonas reinhardtii 의 acetate 및 nicotinic 영양요구체의 연관

홍순주,이호주 한국유전학회 1983 Genes & Genomics Vol.5 No.2

With UV-irradiation, KX812(ac^-) and KX814(nic^-) were isolated, which require the acetate and nicotinamide for normal growth, respectively. Both mutants were able to grow somewhat on the minimal medium upto 10 days and eventually degenerated. Tetrad analysis showed linkages of ac^- to the linkage group XIII and nic^- to the linkage group VI. The gene ac^- has been assigned to the region between 33.1 and 34.4 beyond nic^- 15 on the left arm of the chromosome XIII. The position of nic^- has been mapped between 22.6 and 23.0 on the right arm of the chromosome VI.

Electron Spin Resonance Studies of spin-Trapped Free Radicals Produced by Reaction of Metmyoglobins with Hydrogen Peroxide

홍순주,Hong, Sun-Joo,Piette, Lawrence H. 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

Metmyoglobin을 spin trap 5,5-dimethyl pyrroline-N-oxide (DMPO) 존재 아래 $H_2O_2$반응시켰을 때, 폭이 넓은 다섯 가닥의 비대칭 ESR spectrum을 얻었는데, 기대했던 DMPO/OH adduct의 흔적은 찾을 수 없었다. 또 이 metmyoglobin을 DMPO 존재 아래서 Fenton 시약과 반응시켰을 때도 동일한 결과를 얻었을 뿐 아니라, Fenton 시약과 DMPO를 반응시켜 DMPO-OH adduct 생성을 ESR로 확인한 뒤에 그 용액에 metmyoglobin을 첨가했을 때, 기존의 DMPO-OH signal은 완전히 사라지고 위에서 얻었던 비대칭 다섯가닥 spectrum으로 대치되는 것을 확인했다. 이 결과는 myoglobin 분자의 heme crevice에서 생성된 1 차 및 2 차 라디칼 adduct는 물론, 단백질 분자 표면에서 Fenton 시약에 의해 생성된 DMPO/OH까지도 heme pocket 안에 굳게 고정되는 때문인 것으로 해석했다. 한편 DMPO 대신에 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO)를 사용했더니 DMPO의 경우와는 아주 다르게, 두 종류의 라디칼로 혼성된 ESR spectrum을 얻었는데. 그 중에서 폭넓은 특정 구조가 없는 signal은 TMPO가 단백질 backbone에 있는 ${\alpha}-$ 탄소 라디칼과, 그리고 선명한 세가닥 signal은 단백질의 곁가닥 라디칼과 이룩한 adduct를 각각 나타내는 것으로 해석된다. 그런데 TMPO는 DMPO보다 메틸기를 두 개 더 가지고 있어서 보다 큰 입체적 장애를 받아 pocket에는 들어갈 수가 없고, 단백질 분자의 표면에 생성된 라디칼만을 포착하는 것으로 이해된다. Metmyoglobin when incubated with hydrogen peroxide in the presence of the spin trap, 5,5-dimethyl pyrroline-N-oxide (DMPO) has produced an asymmetric ESR spectrum, consisting of five broad lines of different intensities, in which no evidence of DMPO/OH radical adduct was detected. This result was found to be consistent to other incubation in which metmyoglobin was mixed with Fenton's reagent in the presence of DMPO. Furthermore, DMPO/OH radical adducts validated by the characteristic ESR quartet while incubating Fenton's reagent with DMPO, disappeared completely and was displaced by the asymmetric broad five line signal immediately after addition of metmyoglobin to this incubation solution. These results strongly suggest that not only both of the primary and secondary radical species formed in the vicinity of the heme active site and hence trapped by DMPO, but also additional DMPO/OH radical adducts formed on the surface of the protein molecule by Fenton's reagent/DMPO incubation are together strongly immobilized in the heme pocket of the myoglobin molecule. A similar incubation in which metmyoglobin was mixed with hydrogen peroxide in the presence of the spin trap 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO), however, gave a quite different ESR spectrum with two different signal species, a sharp triplet of equal intensities with a parameter $a_N$ 6.8 G, and a broad, structureless signal covering about 40 G. This indicates that the TMPO, having two more substituents, traps radicals formed on the surface of the protein molecule, in contrast to DMPO, due to the increased steric hindrance and hence can hardly enter into the heme pocket. The broad signal species was ascribed to the TMPO radical adduct formed with ${\alpha}$-carbon atom in backbone and the triplet to that formed with side chains.

어느오후, 골목

洪淳珠,李起鎬 세계평화교수협의회 1983 廣場 Vol.123 No.-

용액에서의 아미노산 및 단백질 자유기에 관한 ESR 연구 제 2 보 Ti - H2O2 flow system 으로 만든 아미노산 자유기의 동정

홍순주,L . H . Piette ( Sun Joo Hong ) 생화학분자생물학회 1971 BMB Reports Vol.4 No.1

Free radicals generated by exposing amino acids to Ti-H₂O₂ flow mixing system were identified by electron spin resonance spectroscopy. Hydrogen atom abstraction by the reactive primary radical, having electrophilic character, was found to occur at the carbon atom furthest from the guanidium, carboxyl and protonated amino group, and sulfur atom. The main radical species from DL-leucine, DL-methionine and L (+) arginine were assigned to be CH₂CH (CH₃) CH₂CH (NH₃^+)COO^-, CH₃SCH₂CHCH(NH₃^+)COO^- and NH=C(NH₂)NHCH₂CHCH₂CH(NH₃^+) COO^- respectively. The splitting for the hydroxyl hydrogen was also clearly observed in the system for DL-serine free radical, CH (OH) CH (NH₃^+) COO^-. Generally, HCl appeared to interfere with the free radical production for most of the amino acids investigated except L-cysteine and DL-methionine in contrast with H₂SO, which enhances most signals but not with the latter.

Chlamydomonas reinhardtii 변이체의 Ribulose - 1 , 5 - Bisphosphate carboxylase 에 관한 연구

홍순주,김영명,이진하,천병익 ( Sun Joo Hong,Young Myeong Kim,Jin Ha Lee,Byoung Ik Cheon ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.2

Ribulose-1, 5-bisphosphate carboxylase isolated from acetate requiring mutants, KX8215 and KX8261, of Chlamydomonas reinhardtii which were obtained by N-methyl-N-nitrosoN-nitrosoguanidine treatment have been characterized. Divalent metal cations, Mg^(2+), Ni^(2+), and Co^(2+) appeared to enchance the enzyme activity whereas halogen anions, Cl^-, I^-, and Br inhibited the enzyme activity. No difference between the mutant enzymes and that of the wild type has been detected in regards of ion effects, pH requirement, and the level of enzyme protein. Optimum temperature of the enzymes from both mutants were found to be higher at 35℃ than at 25℃ of the wild type enzyme. The specific activities of the mutant enzymes were remarkably lower than that of the wild type enzyme whereas Km(RuBP) and Km (CO₂) values of the former were significantly higher than those of the later, indicating that the affinities of the enzyme for RuBP and/or CO₂ are reduced as a result of the mutation. These results seem to support the previous indication that the lowered activities of the mutant enzymes would be due to the alteration in a structural gene for the large subunit in the enzymes and thus the inheritance of the mutation is non-Mendelian.

H2O2 에 의해 생성된 myoglobi 자유라디칼에 대한 ESR 연구

홍순주,Lawrence H . Piette ( Sun Joo Hong,Lawrence H . Piette ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

Metmyoglobin when incubated with hydrogen peroxide in the presence of the spin trap, 5,5-dimethyl pyrroline-N-oxide (DMPO) has produced an asymmetric ESR spectrum, consisting of five broad lines of different intensities, in which no evidence of DMPO/OH radical adduct was detected. This result was found to be consistent to other incubation in which metmyoglobin was mixed with Fenton`s reagent in the presence of DMPO. Furthermore, DMPO/OH radical adducts validated by the characteristic ESR quartet while incubating Fenton`s reagent with DMPO, disappeared completely and was displaced by the asymmetric broad five line signal immediately after addition of metmyoglobin to this incubation solution. These results strongly suggest that not only both of the primary and secondary radical species formed in the vicinity of the heme active site and hence trapped by DMPO, but also additional DMPO/OH radical adducts formed on the surface of the protein molecule by Fenton`s reagent/DMPO incubation are together strongly immobilized in the heme pocket of the myoglobin molecule. A similar incubation in which metmyoglobin was mixed with hydrogen peroxide in the presence of the spin trap 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO), however, gave a quite different ESR spectrum with two different signal species, a sharp triplet of equal intensities with a parameter a_N 6.8 G, and a broad, structureless signal covering about 40 G. This indicates that the TMPO, having two more substituents, traps radicals formed on the surface of the protein molecule, in contrast to DMPO, due to the increased steric hindrance and hence can hardly enter into the heme pocket. The broad signal species was ascribed to the TMPO radical adduct formed with α-carbon atom in backbone and the triplet to that formed with side chains.

학제학문 - 생물물리학 일고

홍순주 한국생화학분자생물학회 (구 한국생화학회) 1985 생화학분자생물학회 소식 Vol.5 No.4

An ESR Study of Amino Acid and Protein Free Radicals in Solution Part Ⅲ. ESR Study of Lysozyme Free Radical Produced by $Ti-H_2O_2$ Flow System

홍순주,Hong, Sun-Joo,Piette, L.H. Korean Chemical Society 1971 대한화학회지 Vol.15 No.4

Free radicals of lysozyme produced by $Ti-H_2O_2$ system were studied in aqueous solution at room temperature using ESR with a continuous flow-mixing. The spectra, each consisting of a doublet with 5.5 G splitting and a broad resonance covering 80 G splitting are closely similar in shape to that for solid irradiated in vacuum at $77^{\circ}K$ and observed at room temperature immediately on warming. The result is assumed to indicate that the secondary protein radical components formed within 0.01 second, dead time of the mixing chamber, and initiated by hydrogen atom abstraction at ${\alpha}$-carbon atom of peptide chain in liquid solution at room temperature are identical to those resulting from the initial formation of a mixture of positive holes and negative ions by ionization processes as well as radical fragments by the rupture of chemical bonds in the solid during similar time at the same temperature. A broad resonance is observed with considerable amplitude on the high field side of the doublet, which is quite dissimilar to the spectra of irradiated solid lysozyme. This resonance was tentatively attributed to the polypeptide free radical in which unpaired electrons are localized on side chain.

Studies on Ribulose-1, 5-Bisphosphate Carboxylase from Acetate Requiring Mutants of Chlamydomonas reinhardtii

홍순주,김영명,이진하,천병익,Hong, Sun-Joo,Kim, Young-Myeong,Lee, Jin-Ha,Cheon, Byoung-Ik 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.3

Chlamydomonas reinhardtii에 N-methyl-N-nitroso-N-nitrosoguanidine을 처리하여 acetate 요구성 변이주, KX8215와 KX8261을 얻었고 여기에서 ribulose-1, 5-bisphosphate carboxylase를 분리하여 그 특성을 연구하였다. 2가 금속이온 $Mg^{2+}$, $Ni^{2+}$, $Co^{2+}$ 등은 효소 활성을 증가시켰고 할로겐족 음이온 $Cl^-$, $I^-$, $Br^-$ 등은 효소 활성을 저해하였으나 $Cu^{2+}$는 활성에 거의 영향을 주지 않았다. 또한 변이주 효소의 이온 영향, 최적 pH 그리고 효소 단백질의 함량 등은 야생종의 경우와 별 차이가 없었다. 변이주 효소의 최적온도는 $35^{\circ}C$로 야생종 효소의 $25^{\circ}C$ 보다 $10^{\circ}C$높았다. 중요한 점은 변이주 효소의 고유 활성도가 야생종 효소보다 현저하게 낮아진데 반해 변이주 효소의 Km(RuBP) 와 Km($Co_2$) 값은 야생종 효소보다 상당히 크게 나타난 것인데 이는 변이의 결과로 효소에 대한 RuBP와 $Co_2$의 친화력이 감소된 때문이라고 생각한다. 이상의 결과는 변이체 효소의 큰 subunit의 구조유전자에 변화가 일어났고 따라서 이 변이의 유전은 non-Mendelian이라는 추리를 가능케 한다. Ribulose-1, 5-bisphosphate carboxylase isolated from acetate requiring mutants, KX8215 and KX8261, of Chlamydomonas reinhardtii which were obtained by N-methyl-N-nitroso-N-nitrosoguanidine treatment have been characterized. Divalent metal cations, $Mg^{2+}$, $Ni^{2+}$, and $Co^{2+}$ appeared to enchance the enzyme activity whereas halogen anions, $Cl^-$, $I^-$, and $Br^-$ inhibited the enzyme activity. No difference between the mutant enzymes and that of the wild type has been detected in regards of ion effects, pH requirement, and the level of enzyme protein. Optimum temperature of the enzymes from both mutants were found to be higher at $35^{\circ}C$ than at $25^{\circ}C$ of the wild type enzyme. The specific activities of the mutant enzymes were remarkably lower than that of the wild type enzyme whereas Km (RuBP) and Km ($Co_2$) values of the former were significantly higher than those of the later, indicating that the affinities of the enzyme for RuBP and/or $Co_2$ are reduced as a result of the mutation. These results seem to support the previous indication that the lowered activities of the mutant enzymes would be due to the alteration in a structural gene for the large subunit in the enzymes and thus the inheritance of the mutation is non-Mendelian.

An ESR Study of Amino Acid and Protein Free Radicals in Solution - PART II. Identification of Amino Acid Free Radicals Produced by Ti-$H_2O_2$ Flow System

홍순주,Hong, Sun-Joo,Piette, L.H. 생화학분자생물학회 1971 한국생화학회지 Vol.4 No.1

Ti-$H_2O_2$ flow-mixing system에서 만들어진 아미노산 자유기를 ESR법으로 동정하고 이 결과와 방사선에 의해 생성된 경우와를 비교 검투했다. 이 system으로 만들어진 각 아미노산의 자유기는 반드시 한 가지 만은 아님이 밝혀졌는데 이 실험에서 얻은 각 ESR 스펙트럼은 glycine에서는 주로 $\dot{C}H({NH_3}^+)COO^-$, DL-$\alpha$-alanine에서는 주로 $\dot{C}H_2CH({NH_3}^+)COO^-$, $\beta$-alanine에서는 주로 $CH_2({NH_3}^+)\dot{C}H{COO_3}^-$, DL-valine에서는 $(CH_3)_2\dot{C}CH({NH_3}^+)COO^-$와 거의 동율로 $\dot{C}H_2CH(CH_3)\dot{C}H({NH_}^+)COO^-$, DL-serine에서는 $\dot{C}H(OH)CH({NH_3}^+)COO^-$, L-cysteine, cystine 및 gIutathione에서는 주로 $RCH_2\dot{S}$, DL-threonine에서는 주로 $CH_3\dot{C}(OH)CH({NH_3}^+)COO^-$, DL-leucine에서는 주로 $\dot{C}H_2CH(CH_3)CH_2CH({NH_3}^+)COO^-$, DL-methionine에서는 주로 $CH_2SCH_2\dot{C}HCH({NH_3}^+)COO^-$, 그리고 L(+)arginine에서는 주로 $HN=C(NH_2)NHCH_2\dot{C}HCH_2CH({NH_3}^+)COO^-$의 생성을 나타내고 있음을 확인했다. 결국, 방사선에 의한 아미노산 자유거의 생성은 알파위치에서 수소원자를 떼어냄으로서 생성되는데 반해 이 system에서는 일차적으로 생성된 친(親)전자성인 수산기가 아미노산 분자의 guanidium 기, carboxyl 기 또는 proton이 더해진 amino 기에서 가급적 먼 위치에서 수소원자를 떼어 냄으로써 생성됨을 알았다. 본실험에서 수산기수소에 의한 splitting이 선명하게 들어난 것은 특기할 일이며 이 실험에서 다룬 아미노산 중에서 L-cysteine과 DL-methionine을 제외한 모든 아미노산의 경우에는 그의 자유기 생성이 염산에 의해 저해를 받은 반면, 황산은 염산의 경우와 정반대의 영향을 끼친다는 사실을 알았다. Free radicals generated by exposing amino acids to Ti-$H_2O_2$ flow mixing system were identified by electron spin resonance spectroscopy. Hydrogen atom abstraction by the reactive primary radical, having electrophilic character, was found to occur at the carbon atom furthest from the guanidium, carboxyl and protonated amino group, and sulfur atom. The main radical species from DL-leucine, DL-methionine. and L(+)arginine were assigned to be $\dot{C}H_2CH(CH_3)CH_2CH({NH_3}^+)COO^-$, $\dot{C}H_3SCH_2\dot{C}HCH({NH_3}^+)COO^-$ and $NH=C(NH_2)NHCH_2\dot{C}HCH_2CH({NH_3}^+)COO^-$ respectively. The splitting for the hydroxyl hydrogen was also clearly observed in the system for DL-serine free radical, $\dot{C}H(OH)CH({NH_3}^+)COO^-$. Generally, HCl appeared to interfere with the free radical production for most of the amino acids investigatd except L-cysteine and DL-methionine in contrast with $H_2SO_4$ which enhances most signals but not with the latter.