소의 이하선에서 $\alpha$-Glycerophosphate 탈수소 효소에 관한 연구

장양섭,이동욱,Chang, Yang-Sup,Lee, Dong-Wook 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.2

소의 이하선 조직에서 세포질과 mitochondria 분획을 분리하고 세포질에 있는 $\alpha$-glycerophosphate 탈수소효소($NAD^+$ 2-oxidoreductase, EC 1.1.1.8) 및 mitochondria에 있는 FAD 의존성 $\alpha$-glycerophosphate 탈수소효소(cytochrome oxidoreductase, EC 1.1.99.5)의 분포를 관찰하였다. 이하선 조직 1 g당 세포질에 있는 NAD 의존성 효소의 활성도는 약 278 unit로 총활성도에 대하여 약 94%가 세포질에 함유되어 있었고 mitochondria에 있는 FAD 의존성 효소의 활성도는 약 16 unit로 총활성도에 대하여 약 44%가 mitochondria에 함유되어 있었다. NAD 의존성 및 FAD 의존성 효소에 의한 $\alpha$-glycerophosphate의 산화 속도가 현저한 차이를 나타내는 점으로 보아 세포질과 mitochondria 구획 사이에서 $\alpha$-glycerophosphate shuttle이 활발히 이루어지지 않는 것 같다. 부분 정제된 NAD 의존성 및 FAD 의존성 효소를 각각 DEAE-cellulose column chromatography를 하였을 때 두 효소 모두 효소의 활성이 있는 두개의 peak가 분리되는 점으로 보아 isozyme이 각각 존재하는 것으로 생각된다. The distribution of NAD-linked $\alpha$-glycerophosphate dehydrogenase ($NAD^+$ 2-oxidoreductase, EC 1.1.1.8) and FAD-linked $\alpha$-glycerophosphate dehydrogenase (cytochrome oxidoreductase, EC 1.1.99.5) of bovine parotid gland have been studied. The activity of NAD-linked $\alpha$-glycerophosphate dehydrogenase was found to be about 278 units per gram tissue and was recovered about 94% in cytosol. FAD-linked $\alpha$-glycerophosphate dehydrogenase activity was about 16 units per gram tissue and the relative activity of mitochondrial fraction was 44%. The oxidation velocity by the NAD- and FAD-linked $\alpha$-glycerophosphate dehydrogenase was strikingly differed between the cytosolic and mitochondrial fraction. This indicates that the $\alpha$-glycerophosphate-dihydroxyacetone phosphate shuttle for the electron transport does not exist in the parotid gland of bovine. In order to identify the isozyme patterns of these enzymes, DEAE-cellulose colum chromatography was performed. The results of this column chromatography showed that these enzymes consist of two activity peaks. This suggests NAD- and FAD-linked $\alpha$-glycerophosphate dehydrogenase of bovine parotid gland consist of several isozymes.

소의 이하선에서 α - Glycerophosphate 탈소소 효소에 관한 연구

장양섭,이동욱 ( Yang Sup Chang,Dong Wook Lee ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.2

The distribution of NAD-linked α-glycerophosphate dehydrogenase (NAD^+ 2-oxidoreductase, EC 1.1.1.8) and FAD-linked α-glycerophosphate dehydrogenase (cytochrome oxidoreductase, EC 1.1.99.5) of bovine parotid gland have been studied. The activity of NAD-linked α-glycerophosphate dehydrogenase was found to be about 278 units per gram tissue and was recovered about 94% in cytosol. FAD-linked α-glycerophosphate dehydrogenase activity was about 16 units per gram tissue and the relative activity of mitochondrial fraction was 44%. The oxidation velocity by the NAD- and FAD-linked α-glycerophosphate dehydrogenase was strikingly differed between the cytosolic and mitochondrial fraction. This indicates that the α-glycerophosphate-dihydroxyacetone phosphate shuttle for the electron transport does not exist in the parotid gland of bovine. In order to identify the isozyme patterns of these enzymes, DEAE-cellulose colum chromatography was performed. The results of this column chromatography showed that these enzymes consist of two activity peaks. This suggests NAD- and FAD-linked α-glycerophosphate dehydrogenase of bovine parotid gland consist of several isozymes.

페닐히드라진으로 유발시킨 토끼 망상적혈구에 있어서 글루탐산 데히드로게나제의 합성과 분해

장양섭,전현우,박재윤 ( Yang Sup Chang,Hyun Woo Jun,Jae Yoon Park ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.2

The biosynthesis and degradation of NAD-linked glutamate dehydrogenase isozymes of red blood cells from phenylhydrazine-treated rabbits have been studied. The results were as follows. The normal red blood cells contained about 4.0 units per ㎖ of red blood cells. The cytosolic isozyme activity was increased significantly after phenylhydrazine treatment and reached a maximum value at the 5th day, that is about 8.6 fold as compared with initial value. The mitochondrial isozyme activity was detected at the 2nd to 3rd day after phenylhydrazine treatment and also attained the peak at the 5th day. Accordingly the increase in total glutamate dehydrogenase activity during the induction of reticulocytosis appeared to be primarily due to the increase in the cytosolic isozyme (K_s=0.1161; K_d=0.0043; t_½=161.16 hours) and partly due to the increase in the activity of mitochondrial isozyme during reticulocytosis (K_s=0.0675; K_d=0.0090; t_½=77.00 hours). The rate constants following recovery from phenylhydrazine treatment were: K_s=0.0916; K_d=0.0229; t_½=30.26 hours for cytosolic isozyme and K_s=0. 0160; K_d=0.0320, t_½=21.66 hours for mitochondrial isozyme.

λPhage 의 PL Promoter 를 갖는 표현형 유전자 운반체의 개발

장양섭,전현우,박재윤 ( Yang Sup Chang,Hyun Woo Jun,Jae Yoon Park ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.2

In this study, series of expression vectors that contain PL-promoter of phage lambda were constructed. The PL-promoters in those plasmids were able to be controlled by temperature in the host Escherichia coli which contained temperature sensitive CI857 gene on the chromosome. Plasmid pPL81, which was constructed with the fragment containing the N gene and its PL-promoter of phage lambda into the BamHI site of pBR322, was digested with ClaI for the deletion of 899 by and self-ligated to construct pPL82. The plasmid pPL82 is useful for constructing gene fusions whose product contained amino acids encoded by N gene. Besides the construction of pPL82, HpaII fragment containing only oLpL region of pPLgl was inserted into the C1aI site of pBR322. The plasmid with the PL promoter in the counterclockwise orientation was named as pPL83, and that in the clockwise orientation was named as pPLg4. These plasmids carry portable promoter that can be separated by codigestion with EcoRI and HindIII restriction endonucleases. Since the β-lactamase genes of pPL81, pPL82 and pPL83 are located at the downstream of the PL-promoter, the β-lactamase can be induced at 42℃. The comparison of the production of β-lactamase indicated that PL-promoter of pPL82 was more efficient than pPL81, and that of pPL83 was the most efficient. These results indicated that when the structural gene is closely located to the PL-promoter, the influence of PL-promoter is more effective.

Biosynthesis and Degradation of L-Glutamate Dehydrogenase Isozymes in Red Blood Cells from Rabbit Treated with Phenylhydrazine

장양섭,전현우,박재윤,Chang, Yang-Sup,Jun, Hyun-Woo,Park, Jae-Yoon 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.2

페닐히드라진을 적용한 토끼에 있어서 망상적혈구가 생성하였다가 소실되는 동안의 글루탐산 데히드로게나제의 활성도의 변화, 즉 이 효소의 세포질 및 미토콘드리아 분획 내의 isozyme의 합성 및 분해 속도를 측정하여 다음과 같은 결론을 얻었다. 1. 정상 토끼의 적혈구에서 이 효소의 총활성도는 4.0 unit/ml RBC이며 미토콘드리아 분획에는 이 효소 활성이 증명되지 않았으므로, 총활성도는 세포질 분획의 isozyme에 의한 것이다. 2. 페닐히드라진으로 유발시킨 망상적혈구의 세포질 분획 isozyme의 활성도는 5일 후에 최고에 달했으며 정상 토끼에 비하여 약 8.6배 증가하였다 ($K_s=0.1161$; $K_d=0.0043$; $t_{1/2}=161.16$ hours). 한편 미토콘드리아의 isozyme 활성도도 이때 최고치에 달하였다($K_s=0.0675$; $K_d=0.0090$; $t_{1/2}=77.00$ hours). 3. 페널히드라진을 적용한 후, 회복기에 있어서 글루탐산 데히드로게나제의 합성 및 분해 속도상수는 다음과 같다. 즉 미토콘드리아에 있어서는 ($K_s=0.0160$; $K_d=0.032$; $t_{1/2}=21.66$ hours)이며 세포질의 isozyme은 ($K_s=0.0916$; $K_d=0.0229$; $t_{1/2}=30.26$ hours)이었다. The biosynthesis and degradation of NAD-linked glutamate dehydrogenase isozymes of red blood cells from phenylhydrazine-treated rabbits have been studied. The results were as follows. The normal red blood cells contained about 4.0 units per ml of red blood cells. The cytosolic isozyme activity was increased significantly after phenylhydrazine treatment and reached a maximum value at the 5th day, that is about 8.6 fold as compared with initial value. The mitochondrial isozyme activity was detected at the 2nd to 3rd day after phenylhydrazine treatment and also attained the peak at the 5th day. Accordingly the increase in total glutamate dehydrogenase activity during the induction of reticulocytosis appeared to be primarily due to the increase in the cytosolic isozyme ($K_s=0.1161$; $K_d=0.0043$; $t_{1/2}=161.16$ hours) and partly due to the increase in the activity of mitochondrial isozyme during reticulocytosis ($K_s=0.0675$; $K_d=0.0090$; $t_{1/2}=77.00$ hours). The rate constants following recovery from phenylhydrazine treatment were: $K_s=0.0916$; $K_d=0.0229$; $t_{1/2}=30.26$ hours for cytosolic isozyme and $K_s=0.0160$; $K_d=0.032$; $t_{1/2}=21.66$ hours for mitochondrial isozyme.

外科的 治療를 加한 肺膿痬 46例에 對하여

張良攝(Yang Sup Chang) 대한외과학회 1967 Annals of Surgical Treatment and Research Vol.9 No.2

Branched - Chain 아미노산 Aminotransferase 에 관한 연구 ( Ⅳ ) 악하선의 Branched - Chain Amino Acid Aminotransferase 에 관하여

이봉제,장양섭,이근배 ( Bong Jae Lee,Ryang Sub Chang,Keun Bai Lee ) 생화학분자생물학회 1980 BMB Reports Vol.13 No.4

The activity and isozyme pattern of branched-chain amino acid aminotromsferase (EC 2. 6. 1. 6) in bull submaxillary gland have been studied, The activity of branched-chain amino acid aminotransferase was measured by the method of Ichihara and Koyama, Isozyme pattern of this enzyme was also examined by using of DEAE-cellulose column chromatography, The results obtained were as follows; 1. In the bull submaxillary gland, the aminotransferase activities for branchedchain amino acids are relatively low compared with those in rat tissues, i, e., intestine, pancreas, and heart, 2. This enzyme was localized in the cytosolic, mitochondrial and nucleic fraction, About 85% of the total activity was found in the cytosolic fraction, 8% in the mitochondrial fraction and 6% in the nucleic fraction, 3. Cytosolic fraction of bull submaxillary gland contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase Enzyme I was eluted by 20mM phosphate buffer from DEAE-cellulose column and catalyzed the transamination of all three branched-chain amino acids, 4. The chromatographic elution profile of the Enzyme I from the bull submaxillary gland was very similar with those of normal rat liver and brain.

Branched-Chain 아미노산 Aminotransferase에 관한 연구(IV) 악하선의 Branched-Chain Amino Acid Aminotransferase에 관하여

이봉재,장양섭,이근배,Lee, Bong-Jae,Chang, Ryang-Sub,Lee, Keun-Bai 생화학분자생물학회 1980 한국생화학회지 Vol.13 No.4

한우의 악하선에 있는 branched-chain아미노산 aminotransferase [EC 2.6.1.6]의 분포 및 isozyme의 pattern을 관찰하였다. 효소의 활성도는 Ichihara and Koyama의 방법으로 측정하였으며 isozyme은 ammonium sulfate에 의한 침전 및 DEAE-cellulose column chromatography에 의하여 분리하였다. 이상과 같이 하여 다음과 같은 결론을 얻었다. 1. 악하선의 branched-chain amino acid aminotransferase 활성도는 흰쥐의 정상조직인 심장, 장관 및 췌장에 비하여 낮았다. 2. BCAT는 세포질, mitochondria 및 핵분획에서 발견되며, 세포질에 약 85%, mitochondria에 약 8%, 핵분획에 약 6%가 분포되어 있었다. 3. 기질에 대한 효소활성도는 L-leucine이 가장 높았다. 4. 세포질에 있는 이 효소를 부분 정제하여 isozyme의 pattern을 관찰하기 위하여 DEAE-cellulose column chromatography를 행한 결과 용출액인 인산염 완충액의 농도가 20mM에서 Enzyme I 만이 검출되었다. 5. 소의 악하선에는 BCAT의 isozyme인 Enzyme II 및 III은 발견할 수 없었다. The activity and isozyme pattern of branched-chain amino acid aminotromsferase (EC 2.6.1.6) in bull submaxillary gland have been studied. The activity of branched-chain amino acid aminotransferase was measured by the method of Ichihara and Koyama. Isozyme pattern of this enzyme was also examined by using of DEAE-cellulose column chromatography. The results obtained were as follows; 1. In the bull submaxillary gland, the aminotransferase activities for branched-chain amino acids are relatively low compared with those in rat tissues, i, e., intestine, pancreas, and heart. 2. This enzyme was localized in the cytosolic, mitochondrial and nucleic fraction. About 85% of the total activity was found in the cytosolic fraction, 8% in the mitochondrial fraction and 6% in the nucleic fraction. 3. Cytosolic fraction of bull submaxillary gland contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase Enzyme I was eluted by 20mM phosphate buffer from DEAE-cellulose column and catalyzed the transamination of all three branched-chain amino acids, 4. The chromatographic elution profile of the Enzyme I from the bull submaxillary gland was very similar with those of normal rat liver and brain.