• Microbiology and Biotechnology Letters
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• v.42 no.1
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• pp.11-17
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• 2014

Selenium is an essential trace element for mammals, but it is very toxic. Therefore, the control of selenium concentrations should be precisely and effectively monitored. Selenium is naturally obtained through foods and seleno-L-methionine (LSeMet) is a major form of selenium. It has been reported that L-SeMet is only converted into Se-adenosyl-L-SeMet. However, a recent study suggested that L-SeMet was directly metabolized into methylselenol ($CH_3SeH$) in mouse liver extract by the reaction of cystathionine ${\gamma}$-lyase (CGL). The canonical reaction of CGL was known to catalyze the cleavage of L-cystathionine to L-cysteine, ${\alpha}$-ketobutyrate and $NH_3$. In the present study, we found that L-SeMet could be directly converted to $CH_3SeH$ using purified homogenous human CGL instead of mouse liver cytosol. Authentic $CH_3SeH$ was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The gaseous product of the enzymatic reaction with L-SeMet was analyzed by GC/MS spectrometry. The GC/MS data was identical to that of authentic dinitrophenyl selenoether. We also analyzed the kinetic parameters for the formation of $CH_3SeH$ from L-SeMet by human and mouse CGL. These results suggest that human CGL is a critical enzyme which is responsible for L-SeMet metabolism.

• Journal of Life Science
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• v.21 no.1
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• pp.119-126
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• 2011

There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$-synthase (CBS) and cystathionine ${\gamma}$-lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.114-126
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• 2019

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine ${\gamma}-lyase$ (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group ($-CH_2CH_2SH$) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

• Journal of Ginseng Research
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• v.36 no.2
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• pp.135-145
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• 2012

Previously, we reported that Helicobacter pylori-associated gastritis and gastric cancer are closely associated with increased levels of hydrogen sulfide ($H_2S$) and that Korean red ginseng significantly reduced the severity of H. pylori-associated gastric diseases by attenuating $H_2S$ generation. Because the incubation of endothelial cells with $H_2S$ has been known to enhance their angiogenic activities, we hypothesized that the amelioration of $H_2S$-induced gastric inflammation or angiogenesis in human umbilical vascular endothelial cells (HUVECs) might explain the preventive effect of Korean red ginseng on H. pylori-associated carcinogenesis. The expression of inflammatory mediators, angiogenic growth factors, and angiogenic activities in the absence or presence of Korean red ginseng extracts (KRGE) were evaluated in HUVECs stimulated with the $H_2S$ generator sodium hydrogen sulfide (NaHS). KRGE efficiently decreased the expression of cystathionine ${\beta}$-synthase and cystathionine ${\gamma}$-lyase, enzymes that are essential for $H_2S$ synthesis. Concomitantly, a significant decrease in the expression of inflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase, and several angiogenic factors, including interleukin (IL)-8, hypoxia inducible factor-1a, vascular endothelial growth factor, IL-6, and matrix metalloproteinases, was observed; all of these factors are normally induced after NaHS. An in vitro angiogenesis assay demonstrated that NaHS significantly increased tube formation in endothelial cells, whereas KRGE pretreatment significantly attenuated tube formation. NaHS activated p38 and Akt, increasing the expression of angiogenic factors and the proliferation of HUVECs, whereas KRGE effectively abrogated this $H_2S$-activated angiogenesis and the increase in inflammatory mediators in vascular endothelial cells. In conclusion, KRGE was able to mitigate $H_2S$-induced angiogenesis, implying that antagonistic action against $H_2S$-induced angiogenesis may be the mechanism underlying the gastric cancer preventive effects of KRGE in H. pylori infection.