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Kinetic Mechanism and End-Product Regulation of Deoxyguanosine kinase from Beef Liver Mitochondria
Park,Inshik,Ives,David H. 東亞大學校附設遺傳工學硏究所 1996 遺傳工學硏究 Vol.- No.3
Initial-rate kinetic measurements with the affinity purified 2-deoxyguanosine (dGuo) kinase from beef liver mitochondria yielded reciprocal plots which converged on the abscissa, with either dGuo or ATP as the varied substrate. The limiting Km for dGuo was 4.7 μM, and that for ATP was 780 μM. One product, dGMP, was competitive with both substrates, while the other, ADP, was competitive with ATP and non-competitive with dGuo. Qualitatively identical results were obtained with an alternative substrate, dTTP, and with alternative product inhibitors, dIMP and dTDP. These results are consistent with a random BiBi kinetic mechanism, judging from the formation of a dead-end complex of the enzyme, dGuo and ADP. dGTP competes very strongly with ATP (Ki=0.03μM), but is non-competitive towards dGuo. The more weakly-bound dGDP is competitive with both substrates.
Park, In-Shik,Ives, David H. Korean Society for Biochemistry and Molecular Biol 2002 Journal of biochemistry and molecular biology Vol.35 No.2
Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased $K_m$ values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements bad no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal end-product inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.
Park, In Shik,Ives, David H . 생화학분자생물학회 1997 BMB Reports Vol.35 No.2
Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased K_m values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.
IKEDA, TAKEHISA,GRIMME, STEPHEN,Ma, NING,IKEDA, SEACHIRO,FIENO, ANGELA,IVES, DAVID H,GUO, SHENYUAN,PARK, INSHIK 東亞大學校附設遺傳工學硏究所 1997 遺傳工學硏究 Vol.- No.4
Three of the four deoxynucleoside kinases required for growth of Lactobacillus acidophilus R-26 exist as heterodimeric pairs specific for deoxyadenosine(dAK) and deoxycytidine (dCK) or dAK and deoxyguanosine (dGK). However, only two tandem genes, dak/dgk, are found, and are expressed only as dAK/dGK in transformed Escherichia coli. Sequencing peptides spanning 63% of the native dCK subunit revealed a sequence identical to that deduced from dgk(beginning MTVIVL…), except that dCK lacks residues 2 and 3 (dCK is M-IVL;dGK is ·TVIVL). Also, mass spectrometry indicates that native dCK and dGK subunits are identical in mass adjusted for the first three residues. Furtheromore, the native enzymes have identical isoelectric pH values, indicating an equal number of charged residues. To enable E. coli to express peptide having the native dCK sequence, codons 2 and 3 were deleted from the dgk portion of the tandem genes, resulting in expression of protein having the specificities and regulatory properties of dAK/dCK, including heterotropic stimulation of dAK activity by deoxycytidine or dCTP (not deoxyguanosine or dGTP) and end-product inhibition of the respective activities by dATP and dCTP. Subcloning normal and mutant dgk yielded homodimeric dGK and dCK, respectively. The dCK homodimer strongly resembles human dCK with a low ?? for deoxycytidine, the ability to phosphorylate deoxyadenosine and deoxyguanosine at much higher ?? values, and end-product inhibition by dCTP. Thus two distinct and specific enzymes evidently are derived from a single Lactobacillus gene. The mechanism by which this occurs in vivo has yet to be elucidated.
Evaluation of Morus alba leaf extracts after bioconversion for development of anti-diabetic agent
Ive Farha MOONMOON,Jin Hyeok KIM,Tae Jung KIM,Kyung Tae KIM,Young Ho KIM,Cheong Won CHO,Hyung Min KIM,Jong Seong KANG 한국분석과학회 2021 학술대회논문집 Vol.2021 No.11
Morus alba has been widely used as herbal medicines and functional foods for its well known bioactivities, such as antitumor, antidiabetic, and antioxidant activities. Recently, many studies have been reported which improved the bioactivities and function of materials using bioconversion technology such as fermentation by microorganisms and enzymes. Therefore, we applied bioconversion method to M. alba to evaluate enhanced bioactivities. The goal of this study was to develop analytical method to evalutate improved bioactivities and to conduct quality control of bio-converted M. alba leaf products. First, liquid-liquid extraction was performed with water and ethyl acetate to achieve best extraction efficiency and to minimize the solvent matrix effects. The water layer was analyzed by HPLC-DAD-ESI-MS on a C18 column (4.6 × 250 mm, 5 ㎛) with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Flow rate was set as 0.7 mL/min with gradient condition, detection with 320 nm of UV detector, and in room temperature. As a result, five peaks were increased upon bioconversion of M. alba leaves. Among increased peaks, four peaks were identified by comparison of UV spectra and molecular weight with the literature and MS spectrum aquired by LC-MS. It was identified as trans-caffeic acid, morin 3-O-ß-D-glucopyranoside, moracin M 3’-O-ß-glucopyranoside, and astragalin. These compounds were well known for bioactivities such as anti-diabetes. Therefore, the increased peaks would help to explain the enhanced bioactivities of enzyme processed M. alba leaves. Collectively, these compounds can be a marker candidates for the quality control of enzyme processed M. alba leaf extracts.
Chong Woon CHO,Farha Ive MOONMOON,Jin Hyeok KIM,Dan GAO,Hyung Min KIM,Jong Seong KANG 한국분석과학회 2021 학술대회논문집 Vol.2021 No.11
Recently, sulfur hexafluoride (SF6) and octafluoropropane (C₃F8) in ophthalmology have been used to treat the retinal detachment by pneumatic retinopexy. However, the fatal medical accidents such as blindness have been reported during the treatment of retinal detachment with these gases. Additionally, these gases as medical gases have no regulation for safety management in each country of the world. The purpose of this study is to develop the analytical method and evaluate the content of main compounds and impurities in these gases for the safety management using GC-BID. GC-BID analysis was carried out on two type columns as follows. One was a capillary column for analyzing carbon dioxide, carbon tetrafluoride, and hexafluoroethane as well as two main compounds. The other was two type of the packed column for analyzing hydrogen, oxygen, nitrogen, methane, and carbon monoxide. The developed method for the impurities and main compounds in sample gases was validated according to ICH guidelines. And, the content of main compounds in two gases were not less than 99.99%. Additionally, the impurities of C₃F8 were hydrogen (< 0.666 ppmv), oxygen (8.2 ppmv) nitrogen (22.7 ppmv), methane (< 0.087 ppmv), carbon monoxide (< 0.126 ppmv) and others (< 20.0 ppmv). While, those of SF6 were oxygen (23.1 ppmv), nitrogen (30.2 ppmv) and others (not more than LODs). The content of all main components and impurities in two gases were suitable in an accordance with inner content criteria. And severe toxic compounds in all two gases were not observed. In conclusion, the content criteria and analytical methods will be useful for the quality control and safety management of impurities and main compound contents in SF6 and C₃F8 samples.