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어피니티 크로마토그라피에 의한 Glucose - 6 - Phosphate Dehydrogenase 의 정제
최양도,변시명,한문희 ( Yang Do Choi,Si Myung Byun,Moon H . Han ) 생화학분자생물학회 1979 BMB Reports Vol.12 No.3
Using the combination procedure of Cibacron Blue F3G-A Sepharose 4B affinity chromatography and hydroxyapatite chromatography, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified rapidly and inexpensively from crude extract of the cell. The Blue-Sepharose affinity chromatography could not separate the two enzymes, glucose-6-phosphate dehydrogenase and lactic dehydrogenase with application of a simple KCl salt gradient. These two enzymes were, however, resolved by the following hydroxyapatite chromatography. The purified enzyme was proved to be homogeneous by polyacrylamide disc gel electrophoresis and SDS gel electrophoresis. The synthesized Blue-Sepharose adsorbent showed dye substitution of 105.94 μmole/g dry gel of 2.27 μmole/㎖ packed gel. The binding capcity of glucose-6-phosphate dehydrogenase was 14.9 U/㎖ packed gel. Blue Dextran 2,000 an analogue of Blue-Sepharose, inhibited the enzyme competitively with respect to NAD^+ and the inhibition constant was determined to be 0.98 μM.
최양도,변시명,한문희,Choi, Yang-Do,Byun, Si-Myung,Han, Moon-H. 생화학분자생물학회 1979 한국생화학회지 Vol.12 No.3
Leuconostoc mesenteroides 에서 추출한 glucose 6-phosphate dehydrogenase를 Cibacron Blue F3GA-Sepharose 4B 어피니티 크로마토그라피와 하이드록시 아파타이트 크로마토그라피 방법에 의해 신속하고 경제적으로 순수 분리하였다. 배양한 세포를 초음파로 파괴하여 얻은 조효소액을 Blue-Sepharose에 어피니티 크로마토그라피한 결과 단순히 KCI gradient만을 사용해서는 glucose-6-phosphate dehydrogenase와 함께 lactate dehydrogenase가 혼합된 부분 정제밖에 얻을 수가 없었다. 그러나 이 두 효소는 다음 단계인 하이드록시 아파타이트 크로마토그라피에 의해 완전히 분리되어 순수한 glucose-6-phosphate dehydrogenase를 얻었다. 이렇게 하여 얻은 glucose-6-phosphate dehydrogenase는 polyacrylamide겔 전기 영동파 SDS겔 전기영동에 의해 그 순수함이 증명되었다. 본 실험을 위해 합성 사용한 Blue-Sepharose 겔의 Cibacron Blue F3G-A 치환도는 건조시킨 겔 1 ml에 대해 $105.94\;{\mu}mole$, 충진된 겔 1 g에 대해 $2.27\;{\mu}mole$을 보여 주었다. 당체가 효소를 결합할 수 있는 능력은 충진 겔 1 ml에 대해 14.9 U이었다. Blue-Sepharose와 유사한 물질인 Blue Dextran 2,000은 효소에 대해 $NAD^+$와 경쟁적 저해 현상을 보였으며 저해상수는 $0.98\;{\mu}M$ 이었다. Using the combination procedure of Cibacron Blue F3G-A Sepharose 4B affinity chromatography and hydroxyapatite chromatography, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified rapidly and inexpensively from crude extract of the cell. The Blue-Sepharose affinity chromatography could not separate the two enzymes, glucose-6-phosphate dehydrogenase and lactic dehydrogenase with application of a simple KCl salt gradient. These two enzymes were, however, resolved by the following hydroxyapatite chromatography. The purified enzyme was proved to be homogeneous by polyacrylamide disc gel electrophoresis and SDS gel electrophoresis. The synthesized Blue-Sepharose adsorbent showed dye substitution of $105.94\;{\mu}mole/g$ dry gel of $2.27\;{\mu}mole/ml$ packed gel. The binding capcity of glucose-6-phosphate dehydrogenase was 14.9 U/ml packed gel. Blue Dextran 2,000 an analogue of Blue-Sepharose, inhibited the enzyme competitively with respect to $NAD^+$ and the inhibition constant was determined to be $0.98\;{\mu}M$.
바실러스 말토제닉 아밀라제 효소 유전자를 이식한 형질전환 감자의 육성 및 특성분석
최양도 서울대학교 농업생명과학대학 농업개발연구소 1998 농업생명과학연구 Vol.2 No.-
By transformation with a gene for maltogenic amylase of Bacillus licheniformis (BLMA), we had attempted to engineer tubers of developing potatoes to produce novel, high value-added carbohydrates. By using two different kinds of promoters such as CaMV promoter for ectopic expression and gbss promoter for tuber specific expression, two different kinds of chimeric recombinants were constructed for potato transformation. PCR, genomic Southern, Northern, BLMA activity and Western analysis results demonstrated that the tansgene was integrated and expressed in transgenic potato plants even though the level of expression was various. Soluble sugar and starch in tubers were analysed by HPIC, which shows evidences that BLMA made an action to modify the tuber starch. Content of short chains in transformed potato starch was decreased to 20-30% that of non-transformed control, which cause to elevate their gelatinization temperature.
Cloning and characterization of jasmonic acid carboxyl methyltransferase genes
Choi, Yang Do 서울대학교 농업개발연구소 2001 농업생명과학연구 Vol.5 No.-
Methyl jasmonate is an important cellular regulator that mediates diverse developmental processes and defense responses. We cloned the novel genes JMT and NTR1 encoding S-adenosyl-L-methionine: jasmonic acid carboxyl methyltransferase from Arabidopsis thaliana and Brassica campestris L. ssp. perkinensis, respectively. Recombinant JMT and NTR1 proteins expressed in E. coli catalyzed the formation of methly jasmonate from jasmonic acid. Kinetic properties, thermal characteristics, optimal pH, and ion-dependency of the NTR1 activity were almost identical to those of Arabidopsis JMT, indicating that these two proteins are orthologues of each other. Transgenic Arabidopsis overexpressing the JMT contained elevated level of endogenous methyl jasmonate, and exhibited constitutive expression of jasmonate-responsive genes and enhanced level of resistance against a virulent fungus. Expression of these genes initiated and limited in developing flower, but propagated systemically, through phloem in vascular bundles, down along with the floral and main stems to reach to the primary root. Moreover, the genes were induced both locally and systemically by wounding or methyl jasmonate treatment. Expression pattern and function of the genes are consistent with previous observations of jasmonate distributions among developing tissues and of jasmonate-responsive defense responses, indicating that the gene activation is a key control point for the jasmonate-regulated responses. Our data suggests that JMT can perceive and respond to local and systemic signals generated during flowering process or by external stimuli, and that the signals may include methyl jasmonate itself. Thus, the gene activation leads to production of methyl jasmonate that could act as a diffusible intra- and inter-cellular signal transducer.