• The Korean Journal of Physiology and Pharmacology
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• 제20권5호
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• pp.449-457
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• 2016

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though $Ca^{2+}$ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ($[Ca^{2+}]_i$) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on $[Ca^{2+}]_i$ in human neutrophils. We observed that NAC ($1{\mu}M{\sim}1mM$) and cysteine ($10{\mu}M{\sim}1mM$) increased $[Ca^{2+}]_i$ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in $[Ca^{2+}]_i$ in human neutrophils was observed. In $Ca^{2+}$-free buffer, NAC- and cysteine-induced $[Ca^{2+}]_i$ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in $[Ca^{2+}]_i$ in human neutrophils occur through $Ca^{2+}$ influx. NAC- and cysteine-induced $[Ca^{2+}]_i$ increase was effectively inhibited by calcium channel inhibitors SKF96365 ($10{\mu}m$) and ruthenium red ($20{\mu}m$). In $Na^+$-free HEPES, both NAC and cysteine induced a marked increase in $[Ca^{2+}]_i$ in human neutrophils, arguing against the possibility that $Na^+$-dependent intracellular uptake of NAC and cysteine is necessary for their $[Ca^{2+}]_i$ increasing activity. Our results show that NAC and cysteine induce $[Ca^{2+}]_i$ increase through $Ca^{2+}$ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

• Molecules and Cells
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• 제26권3호
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• pp.228-235
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• 2008

Thiol-dependent redox systems are involved in regulation of diverse biological processes, such as response to stress, signal transduction, and protein folding. The thiol-based redox control is provided by mechanistically similar, but structurally distinct families of enzymes known as thiol oxidoreductases. Many such enzymes have been characterized, but identities and functions of the entire sets of thiol oxidoreductases in organisms are not known. Extreme sequence and structural divergence makes identification of these proteins difficult. Thiol oxidoreductases contain a redox-active cysteine residue, or its functional analog selenocysteine, in their active sites. Here, we describe computational methods for in silico prediction of thiol oxidoreductases in nucleotide and protein sequence databases and identification of their redox-active cysteines. We discuss different functional categories of cysteine residues, describe methods for discrimination between catalytic and noncatalytic and between redox and non-redox cysteine residues and highlight unique properties of the redox-active cysteines based on evolutionary conservation, secondary and three-dimensional structures, and sporadic replacement of cysteines with catalytically superior selenocysteine residues.

• Journal of Applied Biological Chemistry
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• 제53권4호
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• pp.195-201
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• 2010

B. pseudomycoides로 부터 alanine racemase 유전자를 분리한 다음 이 효소에 존재하는 두개의 cysteine을 하나(C316A) 또는 두개 모두(C316-365A) alanine으로 치환시켰다. 치환된 alanine racemamase는 pET-21 운반체에 삽입한 다음 숙주세포로서 E. coli BL21 (DE3)를 이용하여 발현시켰다. 발현된 단백질은 6XHis이 결합된 affinity chromatography를 이용하여 분리하였으며 SDS-PAGE 분석에서 모두 약 46 kDa의 주요 단일밴드를 나타내었다. Cysteine(-) 변이체의 alanine racemase가 모두 활성도를 보여 cysteine이 catalytic 또는 binding sit에 관여하지 않는 것으로 추정되었다. 변이체 효소들은 wild type에 비하여 열 안정성이 모두 떨어져 $60^{\circ}C$ pH 8.0에서의 활성도 반감시간이 각각 26(wild type), 21(C316A) 18분(C316-365A)-을 나타내었다. 이러한 결과는 cysteine이 열안정화에 상당히 기여함을 알 수 있었다. 그러나 pH 변화에 대한 안정성은 큰 차이가 없었다.

• Journal of Applied Biological Chemistry
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• 제53권3호
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• pp.132-138
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• 2010

B. pseudomycoides로 부터 alanine racemase로 추정되는 유전자를 분리한 다음 6xHistidine 이 결합된 pET-21 운반체를 이용하여 E. coli BL21(DE3)에서 발현시키고 생화학 특성을 조사하였다. 재조합된 alanine racemase는 affinity chromatography를 이용하여 분리하였으며 SDS-PAGE 분석에서 약 46 kDa의 단일밴드를 나타내었다. 분리된 효소는 여러 아미노산 중에서 L-alanine에 대하여 가장 높은 활성도를 보이고 D-alanine에 대하여 두번째로 높은 활성도를 보였다. 따라서 분리된 효소는 alanine racemase로 판단되었다. 분리된 효소는 D-cysteine에 하여 상당히 저해가 되었다. 효소의 최적 활성도는 pH 9.0 근처에서 관찰되었고 산성의 조건보다는 알칼리의 조건에서 보다 안정하였다. 보효소인 PLP 0.3mM의 첨가에 의해 효소의 활성도는 약 70% 가량 증대되었다.

• Parasites, Hosts and Diseases
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• 제43권4호
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• pp.157-160
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• 2005

A 29kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.

• 한국식품저장유통학회지
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• 제20권2호
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• pp.173-181
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• 2013

본 연구는 0.1% 백출 추출물과 0.05% L-cysteine이 양송이버섯의 저장 및 유통 시 갈변억제에 미치는 영향을 조사하기 위하여 각각의 갈변저해제에 양송이버섯을 3분 간 침지 시킨 후 실온에서 1시간 동안 건조시켜 6개씩 PS tray에 담아 PVC랩으로 포장 한 후 저장 기간 별 갈변억제능을 측정하였다. 실험결과 저장 마지막 날 0.1% 백출+0.05% L-cysteine 처리군의 Hunter L 값이 87.24로 가장 높게 유지되었으며, ${\Delta}E$ 값은 5.56으로 색 변화가 적었다. Tyrosinase 저해활성 측정결과 0.1% 백출+0.05% L-cysteine 처리군의 효소저해활성이 높게 측정되어 0.1% 백출 추출물과 0.05% L-cysteine 처리가 갈변억제에 효과적이었다. 또한 0.1% 백출 추출물과 0.05% L-cysteine 병용처리가 양송이버섯의 갈변억제 이외에 품질유지에 어떠한 영향을 끼치는지 조사하기 위하여 품질평가를 시행하였다. 중량감소율과 경도측정 결과 모든 저장일 수에서 대조군보다 0.1% 백출+0.05% L-cysteine 처리군이 중량감소가 적었으며 높은 경도를 유지하였다. 관능평가 결과 전체 기호도 항목에서 대조군이 저장 11일, 0.1% 백출+0.05% L-cysteine 처리군은 14일에 상품성이 상실되어 대조군에 비해 약 3일 가량 상품성이 오래 유지되었다. 본 실험결과를 바탕으로 0.1% 백출 추출물과 0.05% L-cysteine 병용처리는 양송이버섯의 갈변억제에 효과적이며, 중량 및 경도 감소, 이취 발생 등 품질감소를 지연시켜 선도연장에 도움을 줄 것으로 사료된다.

• Journal of Plant Biotechnology
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• 제1권1호
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• pp.13-19
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• 1999

The visco-elastic properties of gluten are major determinants of the processing properties of doughs. These visco-elastic properties are strongly influenced by the ratio of monomeric and polymeric proteins and the size distribution of the polymeric proteins, which make up the gluten fraction of the dough. Recent studies have revealed that other features, such as the number of the cysteine residues of the HMW-GS, also play an important role in determining the functional characteristics. To modify the processing properties at molecular level, the relationship between the structure of molecules and dough properties has to be understood. In order to explore the relationships between individual proteins and dough properties, we have developed procedures for incorporating bacterially expressed proteins into doughs, and measuring their functional properties in small-scale equipment. A major problem in investigating the structure/function relationships of individual seed storage proteins is to obtain sufficient amounts of pure polypeptides from the complex families of proteins expressed in the endosperm. Therefore, we have established a simplified model system in which we produce specific protein genes through bacterial expression and test their functional properties in smallscale apparatus after incorporation into base flour. An S poor protein gene has been chosen as a template gene. This template gene has been modified using standard recombinant DNA techniques in order to test the effects of varying the number and position of cysteine residues, and the size of the protein. Doughs have been mixed in small scale apparatus and characterized with respect to their polymeric composition and their functional properties, including dough mixing, extensibility and small scale bating. We conclude that dough characteristics can be manipulated in a predictable manner by altering the cysteine residues and the size of high molecular weight glutenins.

• Asian-Australasian Journal of Animal Sciences
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• 제30권5호
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• pp.728-735
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• 2017

Objective: This study was performed to reveal the molecular structure and expression patterns of horse glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) genes whose products form glutamate cysteine ligase, which were identified as differentially expressed genes in the previous study. Methods: We performed bioinformatics analyses, and gene expression assay with quantitative polymerase chain reaction (qPCR) for horse GCLC and GCLM genes in muscle and blood leukocytes of Thoroughbred horses Results: Expression of GCLC showed the same pattern in both blood and muscle tissues after exercise. Expression of GCLC increased in the muscle and blood of Thoroughbreds, suggesting a tissue-specific regulatory mechanism for the expression of GCLC. In addition, expression of the GCLM gene increased after exercise in both the blood and muscle of Thoroughbreds. Conclusion: We established the expression patterns of GCLC and GCLM in the skeletal muscle and blood of Thoroughbred horses in response to exercise. Further study is now warranted to uncover the functional importance of these genes in exercise and recovery in racehorses.

• Preventive Nutrition and Food Science
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• 제19권3호
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• pp.220-226
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• 2014

In the present report, the effects of blanching, steaming, and high temperature/high pressure processing (HTHP) on the amino acid contents of commonly consumed Korean root vegetables, leaf vegetables, and pulses were evaluated using an Automatic Amino Acid Analyzer. The total amino acid content of the samples tested was between 3.38 g/100 g dry weight (DW) and 21.32 g/100 g DW in raw vegetables and between 29.36 g/100 g DW and 30.55 g/100 g DW in raw pulses. With HTHP, we observed significant decreases in the lysine and arginine contents of vegetables and the lysine, arginine, and cysteine contents of pulses. Moreover, the amino acid contents of blanched vegetables and steamed pulses were more similar than the amino acid contents of the HTHP vegetables and HTHP pulses. Interestingly, lysine, arginine, and cysteine were more sensitive to HTHP than the other amino acids. Partial Least Squares-Discriminate Analyses were also performed to discriminate the clusters and patterns of amino acids.

• BMB Reports
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• 제43권1호
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• pp.23-28
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• 2010

Binary toxin consisting of BinA and BinB from Bacillus sphaericus is toxic to mosquito larvae. BinB is responsible for specific binding to the larval gut cell membrane while BinA is crucial for toxicity. To investigate functional role of cysteine in BinB, three cysteine residues at positions 67, 161, and 241 were replaced by alanine or serine. Mutations at these positions did not affect protein production and overall structure of BinB. These cysteine residues are not involved in disulfide bond formation between BinB molecules. Mosquito-larvicidal assays revealed that C67 and C161 are essential for toxicity, whereas C241 is not. Mutations at C67 and C161 resulted in weaker BinA-BinB interaction. The loss of toxicity may be due to the reduction of interactions between BinA and BinB or BinB and its receptor. C67 and C161 could also play a part during conformational changes or internalization of the binary toxin into the target cell.