• Biomolecules & Therapeutics
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• v.27 no.2
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• pp.193-200
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• 2019

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z $588.6{\rightarrow}264.4$ for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.174-180
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• 2021

Chalcones exhibit multiple biological activities. Various studies have attempted to modify the structure of chalcones with a special focus on the addition of substituents to the benzene rings. However, these chemical modifications did not improve the water solubility and bioavailability of chalcones. Glycosylation can markedly affect the physical and chemical properties of hydrophobic compounds. Here, we evaluated the ability of a highly promiscuous glycosyltransferase (GT) BsGT1 from Bacillus subtilis ATCC 6633 to biosynthesize chalcone glucosides. Purified BsGT1 catalyzed the conversion of 4'-hydroxychalcone (compound 1), 4'-hydroxy-4-methylchalcone (compound 2), and 4-hydroxy-4'-methoxychalcone (compound 3), into chalcone 4'-O-β-D-glucoside (compound 1a), 4-methylchalcone 4'-O-β-D-glucoside (compound 2a), and 4'-methoxychalcone 4-O-β-D-glucoside (compound 3a), respectively. To avoid the addition of expensive uridine diphosphate glucose (UDP-Glc), a whole-cell biotransformation system was employed to provide a natural intracellular environment for in situ co-factor regeneration. The yields of compounds 1a, 2a, and 3a were as high as 90.38%, 100% and 74.79%, respectively. The successful co-expression of BsGT1 with phosphoglucomutase (PGM) and UDP-Glc pyrophosphorylase (GalU), which are involved in the biosynthetic pathway of UDP-Glc, further improved the conversion rates of chalcones (the yields of compounds 1a and 3a increased by approximately 10%). In conclusion, we demonstrated an effective whole-cell biocatalytic system for the enzymatic biosynthesis of chalcone β-D-glucoside derivatives.

• Journal of Life Science
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• v.24 no.1
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• pp.86-91
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• 2014

Trichoplusia ni cells are used as a host permissive cell line in the baculovirus expression system, which is useful for large-scale production of human sugar transport proteins. However, the activity of endogenous sugar transport systems in insect cells is extremely high. Therefore, the transport activity resulting from the expression of exogenous transporters is difficult to detect. Furthermore, very little is known about the nature of endogenous insect transporters. To exploit the expression system further, the effect of D-fructose on 2-deoxy-D-glucose (2dGlc) transport by T. ni cells was investigated, and T. ni cell-expressed human transporters were photolabeled with [$^3H$] cytochalasin B to develop a convenient method for measuring the biological activity of insect cell-expressed transporters. The uptake of 1 mM 2dGlc by uninfected- and recombinant AcMPV-GTL infected cells was examined in the presence and absence of 300 mM of D-fructose, with and without $20{\mu}M$ of cytochalasin B. The sugar uptake in the uninfected cells was strongly inhibited by fructose but only poorly inhibited by cytochalasin B. Interestingly, the AcMPV-GTL-infected cells showed an essentially identical pattern of transport inhibition, and the rate of 2dGlc uptake was somewhat less than that seen in the non-infected cells. In addition, a sharply labeled peak was produced only in the AcMPV-GTL-infected membranes labeled with [$^3H$] cytochalasin B in the presence of L-glucose. No peak of labeling was seen in the membranes prepared from the uninfected cells. Furthermore, photolabeling of the expressed protein was completely inhibited by the presence of D-glucose, demonstrating the stereoselectivity of labeling.

• Applied Biological Chemistry
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• v.27 no.2
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• pp.119-128
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• 1984

The unsaponifiable from rice bran oil was fractionated into 4-desmethyl-'4-monomethyl- and 4, 4-dimethylsterol (triterpene alcohol) fraction by thin layer chromatography (TLC), and sterol composition of the each fraction was analyzed by gas liquid chromatography(GLC). The sterol peaks not well separated by GLC were further fractionated by $AgNO_3-TLC$, then analyzed using GLC. Each components in the three sterol fractions were identified by GLC and gas chromatography-maps spectrometry. As the results, ten sterols were confirmed as 4-desmethylsterol, nine as 4-monomethylsterol and four as 4, 4-dimethylsterol. Such uncommon phytosterols in higher plants as fucosterol, 24-ethyllophenol, 4${\alpha}$-methylstigmasta-7, 25-dienol and 28-isocitrostadienol were detected in rice bran oil and the biosynthetic pathways of the phytosterols were deduced with all the identified sterols.

• Korean Journal of Food Science and Technology
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• v.14 no.3
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• pp.226-231
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• 1982

In order to define triglyceride compositions in fat and oil triglycerides were separated by thin layer chromatography (TLC) from corn oil, and the separated triglycerides were graduated according to each partition number(PN) by high performance liquid chromatography (HPLC) using column of ${\mu}-Bondapack\;C_{18}$ and each graduation was graduated again according to acylcarbon number by gas liquid chromatography(GLC). Fatty acid compositions were analyzed by GLC after their graduation were methylated in according to PN. The triglyceride compositions were estimated by synthesizing the above three results. The estimated triglycerides consisted of 36 kinds in corn oil. The major triglyceride compositions of sample oil were as follows: 21.5%$(C_{18:2},\;C_{18:2},\;C_{18:1})$, 17.4%$(C_{18:1},\;C_{18:2},\;C_{18:1})$, 15.4%$(C_{18:1},\;C_{18:2},\;C_{16:0})$, 11.1%$(C_{16:0},\;C_{18:2},\;C_{18:2})$, 9.0%$(C_{18:1},\;C_{18:1},\;C_{18:1})$, 8.0%$(C_{18:2},\;C_18:2},\;C_{18:2})$, 5.7%$(C_{18:1},\;C_{18:1},\;C_{16:0})$, 2.2%$(C_{16:0},\;C_{16:0},\;C_{18:2})$, 1.6%$(C_{18:2},\;C_{18:2},\;C_{18:2})$, 1.1%$C_{18:2},\;C_{18:0},\;C_{16:0})$, 1.1%$(C_{16:0},\;C_{16:0},\;C_{18:1})$.

• Korean Journal of Food Science and Technology
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• v.14 no.3
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• pp.219-225
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• 1982

Triglycerides of cottonseed oil were separated by thin layer chromatography (TLC), and fractionated by high-performance liquid chromatography (HPLC) on the basis of partition numbers. From each fraction, it was fractionated again on the basis of acyl carbon numbers using gas liquid chromatography (GLC). The fatty acids of triglyceride for each partition number group were analyzed by GLC. From, these results, triglyceride constituents of cotton seed oil were estimated to be 37 kinds of triglycerides. The major triglycerides and their contents in cotton seed oil were as follows: 25.8%$(C_{16:0},\;C_{18:2},\;C_{18:2})$, 15.5%$(C_{18:2},\;C_{18:2},\;C_{18:2})$, 13.8%$(C_{16:0},\;C_{18:2},\;C_{16:0})$, 8.3%$(C_{18:2},\;C_{18:1},\;C_{18:2})$, 6.2%$(C_{18:2},\;C_{18:1},\;C_{18:1})$, 4.1%$(C_{18:1},\;C_{18:1},\;C_{14:0})$, 3.4%$(C_{16:0},\;C_{18:1},\;C_{16:0})$, 2.3%$(C_{18:1},\;C_{18:2},\;C_{16:0})$, 2.2%$(C_{18:1},\;C_{18:1},\;C_{18:1})$, 1.0%$(C_{14:0},\;C_{18:2},\;C_{18:1})$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.11
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• pp.1647-1653
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• 2010

Glycosaminoglycan (GAG) was purified from polysaccharide extracted from sea hare muscle on DEAE-Sepharose column and investigated for the functional groups, distribution of sugars, composition of disaccharide and structure of GAG. Purified GAG was composed of disaccharide above 55% of total sugar. Purified GAG showed amide I peak in 1648/cm and C-O stretch peak as properties of carbohydrate, amino acid peak in 1457/cm, and peak in 866/cm as properties of monosaccharide by FT-IR. Fucose, N-acetylgalactosamine, N-acetylglucosamine, glucose, galactose, mannose and xylose were found in MALDI-TOF MS/MS spectra of hydrolysates by chondroitin sulfate ABC lyase and heparanase I. Purified GAG was expected to be heparan sulfate including N-acetylgalactosamine and N-acetylglucosamine above 70% of total sugar. The structure of GAG was supposed as GlyUA(2S)-GlcNS and GlyUA-GlcNS(6S) with O-linkage on protein core.

• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.1011-1017
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• 2010

A novel dioscin-glycosidase that specifically hydrolyzes multi-glycosides, such as 3-O-${\alpha}$-L-($1{\to}4$)-rhamnoside, 3-O-${\alpha}$-L-($1{\to}2$)-rhamnoside, 3-O-${\alpha}$-L-($1{\to}4$)-arabinoside, and ${\beta}$-D-glucoside, on diosgenin was isolated from the Absidia sp.d38 strain, purified, and characterized. The molecular mass of the new dioscin-glycosidase is about 55 kDa based on SDS-PAGE. The dioscin-glycosidase gradually hydrolyzes either 3-O-${\alpha}$-L-($1{\to}4$)-Rha or 3-O-${\alpha}$-L-($1{\to}2$)-Rha from dioscin into 3-O-${\alpha}$-L-Rha-${\beta}$-D-Glc-diosgenin, further rapidly hydrolyzes the other ${\alpha}$-L-Rha from 3-O-${\alpha}$-L-Rha-${\beta}$-D-Glc-diosgenin into the main intermediate products of 3-O-${\beta}$-D-Glc-diosgenin, and subsequently hydrolyzes these intermediate products into aglycone as the final product. The enzyme also gradually hydrolyzes 3-O-${\alpha}$-L-($1{\to}4$)-arabinoside, 3-O-${\alpha}$-L-($1{\to}2$)-rhamnoside, and ${\beta}$-D-glucoside from [3-O-${\alpha}$-L-($1{\to}4$)-Ara, 3-O-${\alpha}$-L-($1{\to}4$)-Rha]-${\beta}$-D-Glc-diosgenin into diosgenin as the final product, exhibiting significant differences from previously reported glycosidases. The optimal temperature and pH for the new dioscin-glycosidase is $40^{\circ}C$ and 5.0, respectively. Whereas the activity of the new dioscin-glycosidase was not affected by $Na^+$, $K^+$, and $Mg^{2+}$ ions, it was significantly inhibited by $Cu^{2+}$ and $Hg^{2+}$ ions, and slightly affected by $Ca^{2+}$ ions.

• Applied Biological Chemistry
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• v.27 no.4
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• pp.278-284
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• 1984

The triglyceride compositions of peach kernel and apricot kernel oil have been investigated by a combination of high performance liquid chromatography (HPLC) and gas liquid chromatography(GLC). The triglycerides of peach kernel and apricot kernel oil were first separated by thin layer chromatography(TLC), and fractionated on the basis of their partition number(PN) by HPLC on a C-18 ${\mu}-Bondapak$ column with methanol-chloroform solvent mixture. Each of these fractionated groups was purely collected and analyzed by GLC according to acyl carbon number(CN) of triglyceride. Also the fatty acid compositions of these triglycerides were determined by GLC. From the consecutive analyses of these three chromatography techniques, the possible triglyceride compositions of peach kernel and apricot kernel oil were combinated into fifteen and thirteen kinds of triglycerides, respectively. The major triglycerides of peach ternel oil were those of $(3{\times}C_{18:1}\;30.9%)$, $(2{\times}C_{18:1},\;C_{18:2},\;21.2%)$, $(C_{18:1},\;2{\times}C_{18:2}\;10.6%)$, $(3{\times}C_{18:2}\;3.8%)$, $(C_{18:0},\;2{\times}C_{18:1},\;1.8%)$, $(C_{16:0},\;C_{18:1},\;C_{18:2},\;1.5%)$, $(C_{18:0},\;C_{18:1},\;C_{18:2},\;1.1%)$ and those of apricot kernel oil were $(3{\times}C_{18:1},\;39.5%)$, $(2{\times}C_{18:1},\;C_{18:2},\;24.5%)$, $(C_{18:0},\;2{\times}C_{18:2},\;14.2%)$, $(3{\times}C_{18:2},\;2.0%)$.

• Journal of Nutrition and Health
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• v.14 no.1
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• pp.16-25
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• 1981

Commercially available duck-meat was subjected to proximate analysis. On a wet basis, the duck-meat contained 62.87, 17.05, 19.06 ana 1.02 percent of moisture, crude fat, crude protein and ash, respectively. Almost all the essential amino acids contained in the duck-meat protein, ana the tryptophan was the limiting one by amino acid analysis of GLC. An analysis of the fatty acid composition by GLC showed a relatively high concentration of oleic acid. There was also a considerable content of linoleic acid. The content of polyunsaturated fatty acids of duck-meat was 70.9% and the P/S ratio of fatty acids was 3.4. The cholesterol content in duck-meat was determined to be approximately 70. 5mg/100g ofm sample. According to blood analysis, it was understood that the content of phospholipids was relatively high, particulary in lecithin. ATP-phosphorus, at the higher temperature, was released faster than at the lower temperature, by two hours after postmortem. The ATPase activity of Myogibril was inhibited at the relatively high concentration of added EDTA and metallic ions, but the activity was very high in the lower concentrations. According to the cooking conditions, boiled duck-meat showed good digestion by pepsin. It was understood that the digestibility of duck meat was relatively high, so the duck-meat protein is good source of animal protein. Therefore, it is able to be recommended that duck-meat is good nitrogen source animal food.