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Phee, Bong-Kwan,Shin, Dong Ho,Cho, Jin-Hwan,Kim, Seong-Hee,Kim, Jeong-Il,Lee, Youn-Hyung,Jeon, Jong-Seong,Bhoo, Seong Hee,Hahn, Tae-Ryong WILEY-VCH 2006 Proteomics Vol. No.
<P>Phytochrome-interacting proteins have been extensively studied to elucidate light-signaling pathway in plants. However, most of these proteins have been identified by yeast two-hybrid screening using the C-terminal domain of phytochromes. We used co-immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N-terminal domain as well as C-terminal domain. A total of 16 protein candidates were identified, and were selected from 2-DE experiments. Using MALDI-TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A-interacting proteins and the remaining ones to be phytochrome B-interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66-kDa protein were strong candidates as novel phytochrome-interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light-signaling functions. A transgenic knockout Arabidopsis study showed that a 66-kDa protein candidate regulates hypocotyl elongation in a light-specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light-specific changes in hypocotyl elongation. These results suggest that co-immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein-protein interactions in the cell.</P>
Purification and Characterization of Chloroplast Fructose-1,6-bisphosphatase from Pea Leaves
부성희,한태룡,Bhoo, Seong-Hee,Hahn, Tae-Ryong 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.4
엽록체 fructose-1,6-bisphosphatase를 완두 잎으로부터 1,300배 정제하였다. 정제된 효소는 거의 순수하였고 SDS polyacrylamide 전기영동하여 얻은 monomer의 분자량은 약 40,000이었다. 효소는 pH 7.5에서 활성이 없었으나 dithiothreitol 혹은 알카리 pH에서 활성화가 되었다. 이 결과는 분리된 fructose-1,6-bisphosphatase가 엽록체로부터 얻어진 것임을 나타낸다. Fructose-1,6-phosphate와 $Mg^{++}$에 대한 효소 포회곡선은 Hill 상수 2.6 및 3.1을 가진 sigmoidal한 모양을 보여주는데 이는 효소가 multimer로 구성된 allosteric 한 것임을 알 수 있다. 최고 활성의 반에 요구되는 기질농도는 $40{\mu}M$로서 이는 시금치 엽록체 효소에 대한 $80{\mu}M$값 (Zimmermann et al., 1976)보다 작았다. Chloroplast fructose-1,6-bisphosphatase was purified 1,300 fold from pea leaves. The purified enzyme appeared homogeneous and the approximate molecular weight of the monomer was 40,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was inactive at pH 7.5, while it was activated by dithiothreitol or alkaline pH, indicating that the purified fructose-1,6-bisphosphatase was originated from chloroplast. The enzyme saturation curves with fructose-1,6-bisphosphate and $Mg^{++}$ show sigmoidal shapes with almost same Hill coefficients (2.6 and 3.1, respectively), suggesting that the enzyme is composed of multimer. The substrate concentration required for half-maximal activity was $40{\mu}M$ which is a comparable value $(80{\mu}M)$ for spinach chloroplast fructose bisphosphatase (Zimmermann et al., 1976).
완두엽록체 fructose - 1 , 6 - bisphosphatase 의 분리정제 및 특성
부성희,한태룡 ( Seong Hee Bhoo,Tae Ryong Hahn ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.4
Chloroplast fructose-1,6-bisphosphatase was purified 1,300 fold from pea leaves. The purified enzyme appeared homogeneous and the approximate molecular weight of the monomer was 40,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was inactive at pH 7.5, while it was activated by dithiothreitol or alkaline pH, indicating that the purified fructose-1,6-bisphosphatase was originated from chloroplast. The enzyme saturation curves with fructose-1,6-bisphosphate and Mg^(++) show sigmoidal shapes with almost same Hill coefficients (2.6 and 3.1, respectively), suggesting that the enzyme is composed of multimer. The substrate concentration required for half-maximal activity was 40 μM which is a comparable value (80μM) for spinach chloroplast fructose bisphosphatase (Zimmermann et al., 1976).
Biochemical characterization of a regiospecific flavonoid 3'-O-methyltransferase from orange
Park Hye Lin,Bhoo Seong Hee,Lee Sang-Won,Cho Man-Ho 한국응용생명화학회 2024 Applied Biological Chemistry (Appl Biol Chem) Vol.67 No.-
Citrus plants have diverse methoxyflavonoids including, chrysoeriol, isosakuranetin, and nobiletin. In plants, O-methyltransferases (OMTs) participate in the methylation of a vast array of secondary metabolites, including flavonoids, phenylpropanoids, and alkaloids. To identify functional OMTs involved in the formation of methoxyflavonoids, orange (Citrus sinensis) OMT (CsOMT) genes were retrieved from the Citrus Genome Database. The phylogenetic relationships with functional OMTs suggested that three CsOMTs, CsOMT15, CsOMT16, and CsOMT30, are possible candidates for flavonoid OMTs (FOMTs). These CsOMTs were heterologously expressed in Escherichia coli, and their OMT activity was examined with flavonoid substrates. Of the examined CsOMTs, CsOMT16 catalyzed the regiospecific 3’-O-methylation of flavonoids to the respective 3’-methoxyflavonoids. A kinetic study demonstrated that CsOMT16 accepts diverse flavonoids as a substrate with a comparable preference. The flavonoids eriodictyol, luteolin, and quercetin were efficiently converted to homoeriodictyol, chrysoeriol, and isorhamnetin by CsOMT16- transformed E. coli cells, respectively. These findings suggest that CsOMT16 contributes to the methoxyflavonoid formation in orange and is applicable to the biotechnological production of 3’-methoxyflavonoids.
( Jin Hwan Cho ),( Hee Youn Hwang ),( Man Ho Cho ),( Yong Kook Kwon ),( Jong Seong Jeon ),( Seong Hee Bhoo ),( Tae Ryong Hahn ) 한국식물학회 2008 Journal of Plant Biology Vol.51 No.4
Rubisco is a major photosynthetic plant enzyme in the chloroplasts, catalyzing a photosynthetic reaction through carboxylation and oxygenation in the leaves. Despite its biological importance, its high abundance causes difficulties in the proper separation of protein mixtures during 2-dimensional gel electrophoresis (2-DE). Here, we resolved those plant soluble proteins by efficiently removing Rubisco. This resulted in a high quality and resolution of 2-DE gels. Rubisco removal was achieved through aggregation in the presence of a high DTT concentration, which subsequently increased the visualization of less abundant proteins and reduced horizontal streaking. This simple method may provide a means for finding more biologically important protein targets via plant proteomics.