• Journal of Life Science
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• v.22 no.6
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• pp.778-787
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• 2012

The influence of salicylic acid (SA) on growth, chlorophyll, and rubisco/rubisco activase and effect of denaturator on rubisco/rubisco activase activity were studied in tobacco plants grown in vitro with cadmium (Cd) treatment. In order to find out the optimum concentration of SA, tobacco plants treated with $10^{-6}$ mM - $10^2$ mM of SA were grown in MS medium for 9 weeks, respectively. The most pronounced effect on in vitro growth was found at $10^{-4}$ mM SA. Among the control (not treated with Cd and SA), SA, Cd, and Cd + SA, the growth and content of chlorophyll were in the sequence of Cd < Cd + SA < control < SA, and significantly higher at SA compared with others. Similar results were also observed in the content and activity of rubisco and rubisco activase. These data suggest that inhibitory effect by Cd was reversed by SA. These results also indicate that SA has a positive effect on Cd. The effect of denaturants on rubisco activity showed in the sequence of Cd < Cd + SA < control < SA. Rubisco activity was promoted by L-cysteine and ${\beta}$-mercaptoethanol, not by urea, thiourea, and guanidium-HCl. These data suggest that urea, thiourea, and guanidium-HCl are able to act as denaturator, and L-cysteine and ${\beta}$-mercaptoethanol are not. None of the five denaturants affected the activity of rubisco activase.

• Journal of the Korean Magnetic Resonance Society
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• v.16 no.1
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• pp.34-45
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• 2012

Melanocortin-4 receptor (MC4R) subtype is associated with obese humans. Especially, in a patient with severe early-onset obesity, novel heterozygous mutation in the MC4R gene was detected, resulting in an exchange of aspartic acid to asparagine in $90^{th}$ amino acid residue located in the predicted second trans-membrane domain (TM2). Mutations in the melanocortin-4 receptor (MC4R) gene are the most frequent monogenic causes of severe obesity which have been described as heterozygous with loss of function. In order to compare structure difference between MC4R wild type (MC4R-TM2-wt) and mutant (MC4R-TM2-D90N), we designed both MC4R-TM2-wt and MC4R-TM2-D90N construct in pET 21b vector. In this study, we optimized high-yield purification procedure for recombinant TM2-D90N. Eluted recombinant protein was resolubilized under urea condition for thrombin cleavage reaction and we conducted the high-performance liquid chromatography (HPLC) with reverse phase column under 1% acetonitrile, 0.01% TFA buffer solution. The molecular size of purified target peptide was confirmed by Tricine-SDS page analysis. To characterize MC4R-TM2-D90N, we have performed $^{15}N$-isotope labeling of peptide using M9 media and purified labeled target peptide for hetero-nuclear NMR spectroscopy.