• BMB Reports
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• 제45권3호
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• pp.147-152
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• 2012

Methylglyoxal (MG) was identified as an intermediate in non-enzymatic glycation and increased levels were reported in patients with diabetes. In this study, we evaluated the effects of MG on the modification of ferritin. When ferritin was incubated with MG, covalent crosslinking of the protein increased in a time- and MG dose-dependent manner. Reactive oxygen species (ROS) scavengers, $N-acetyl-_L-cysteine$ and thiourea suppressed the MG-mediated ferritin modification. The formation of dityrosine was observed in MG-mediated ferritin aggregates and ROS scavengers inhibited the formation of dityrosine. During the reaction between ferritin and MG, the generation of ROS was increased as a function of incubation time. These results suggest that ROS may play a role in the modification of ferritin by MG. The reaction between ferritin and MG led to the release of iron ions from the protein. Ferritin exposure to MG resulted in a loss of arginine, histidine and lysine residues. It was assumed that oxidative damage to ferritin caused by MG may induce an increase in the iron content in cells, which is deleterious to cells. This mechanism, in part, may provide an explanation or the deterioration of organs under diabetic conditions.

• Bulletin of the Korean Chemical Society
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• 제33권9호
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• pp.3048-3054
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• 2012

The use of aspirin is widely recommended for the prevention of heart attacks owing to its ability to inhibit platelet activation by irreversibly blocking cyclooxygenase 1. However, aspirin also affects the fibrinolytic and hemostatic pathways by mechanisms that are not well understood, causing severe hemorrhagic complications. Here, we investigated the ability of aspirin and aspirin metabolites to inhibit thrombin-activatable fibrinolysis inhibitor (TAFI), the major inhibitor of plasma fibrinolysis. TAFI is activated via proteolytic cleavage by the thrombin-thrombomodulin complex to TAFIa, a carboxypeptidase B-like enzyme. TAFIa modulates fibrinolysis by removing the C-terminal arginine and lysine residues from partially degraded fibrin, which in turn inhibits the binding of plasminogen to fibrin clots. Aspirin and its major metabolites, salicylic acid, gentisic acid, and salicyluric acid, inhibit TAFIa carboxypeptidase activity. Salicyluric acid effectively blocks activation of TAFI by thrombin-thrombomodulin; however, salicylates do not inhibit carboxypeptidase N or pancreatic carboxypeptidase B. Aspirin and other salicylates accelerated the dissolution of fibrin clots and reduced thrombus formation in an in vitro model of fibrinolysis. Inhibition of TAFI represents a novel hemostatic mechanism that contributes to aspirin's therapy-associated antithrombotic activity and hemorrhagic complications.

• BMB Reports
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• 제28권2호
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• pp.138-142
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• 1995

An anticoagulant/fibrinolytic protease was purified to homogeneity from the earthworm Lumbricus rubellus. The protein was a single chain glycoprotein of 32 kDa that exhibited strong proteolytic activity on human thrombin and fibrin clots. Proteolytic degradation of these plasma proteins by the purified enzyme occurred at a neutral pH range. Among several human plasma proteins tested as possible substrates for the protease reaction, the 32 kDa enzyme specifically hydrolyzed both thrombin and fibrin polymers without affecting other proteins, such as serum albumin, immunoglobulin, and hemoglobin. Treatment of the purified enzyme at neutral pH with either phenylmethylsulfonylfluoride or soybean trypsin inhibitor resulted in a loss of catalytic activity. The enzyme hydrolyzed the chromogenic substrate H-D-Phe-L-Pipecolyl-L-Arg-p-nitroanilide with a $K_m$ value of 1.1 ${\mu}M$ at a neutral pH. These results suggest that the anticoagulant/fibrinolytic enzyme from Lumbricus rubellus is a member of the serine protease family having a trypsin-like active site, and one of the potential clevage sites for the enzyme is the carbonyl side of arginine residues in polypeptide chains.

• The Plant Pathology Journal
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• 제20권4호
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• pp.247-251
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• 2004

We developed a polyclonal antibody (PAh) based- ELISA system to accurately and rapidly monitor inocula on plant surface before onset of anthracnose. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides was determined by using indirect ELISA. It was high enough to be detectable up to ${\times}$ 12,800 dilutions. Absorbance readings exceeded (1.5even at a 10$^{-5}$ dilution. Sensitivity of PAb was precise enough to detect spore concentration as low as 50 conidia/well by indirect ELISA. PAb1 and PAb2 proved to be very sensitive and highly specific to the target pathogen, C. gloeosporioides, apparently discriminating other unrelated pathogens, or epiphytes. Absorbance values for original isolate exceeded 1.0, but no reaction was detected with other isolates, except three other anthracnose fungi: C. gloeosporioides (pepper strain), Glomerella cingulata (apple strain) and C. lagenarium. Our data suggest that PAb1 and PAb2 bind with the protein epitope that partially contains residues of amino acid, arginine, and Iysine. This kit fulfills the require-ments for detecting inoculums before infection and during onset of anthracnose on sweet persimmon.

• 약학회지
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• 제43권1호
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• pp.68-76
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• 1999

Phenoxazinone synthase catalyzes the oxidative condensation of two molecules of substituted o-aminophenol to the phenoxazinone chromophore of actinomycin. Mutant strain, Streptomyces sp. V-8-M-1 producing higher phenoxazinone synthase, was obtained from Streptomyces sp. V-8 by treatment of N-methyl-N'-nitro-N-nitrosoguanidine. The phenoxazinone synthase was purified from extract of mutant strain of Streptomyces sp. V-8-M-l by successive steps of streptomycin sulfate, ammonium sulfate precipitation. DEAE-cellulose and Sephadex G-200 column chromatography. Molecular weight of the enzyme was 360,000 daltons. The enzyme was composed of octamer of a single subunit of 45,000 daltons. The Km value and Vmax value for 3-HAA were $14.9{\;}{\mu}M$ and 9.5 mg/U, respectively. The optimal pH and temperature for the enzyme activity were 9.0 and $25~30^{\circ}C$, respectively. Treatment of the enzyme with group specific reagents, phenylglyoxal, p-hydroxymercury-benzoate, Nbromosuccinimide, 5.5'-dithiobis-nitrobenzoic acid and ethylmaleimide resulted in loss of enzyme activity, which shows arginine and cysteine residues are at or near the active site.

• Mycobiology
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• 제36권1호
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• pp.50-54
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• 2008

This study examined the chemical composition of A. blasiliensis and the chemical structural properties of an immuno-stimulating polysaccharide. The amino acids, free sugars, and organic acids by HPLC and fatty acids by GC were analyzed. The immuno-stimulating substance from A. blasiliensis was extracted with hot water and purified by ethanol precipitation. It underwent ion exchange chromatography on DEAE-cellulose and gel filtration on Toyopearl HW 65F. Through GP-HPLC, the substance was found to be homogeneous. Its chemical structure was determined by $^{13}C-NMR$. Fatty acids, organic acids, and sugar alcohol composition consisted exclusively of linoleic acid, fumaric acid and mannitol, respectively. The amino acids were mainly glutamic acid, glycine, and arginine. By $^{13}C-NMR$ analysis, the immuno-stimulating substance was identified as ${\beta}-(1{\rightarrow}3)\;(1{\rightarrow}6)$-glucan, composed of a backbone with $(1{\rightarrow}3)$-linked D-glucopyranosyl residues branching a $(1{\rightarrow}6)$-linked D-glucopyranosyl residue. The ${\beta}$-glucan from A. blasiliensis showed pronounced immuno-stimulating activity on the antibody-production ability of B-lymphocytes by the hemolytic suspension assay. In these results, A. blasiliensis was estimated to have potent pharmacological properties and potential nutritional values.

• Archives of Pharmacal Research
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• 제24권4호
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• pp.355-359
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• 2001

A novel HPLC method with electrochemical detection has been developed for the determination of recombinant human epidermal growth factor (rhEGF) in pharmaceutical products. rhEGF was separated from other components in formulation on a reversed-phase C18 column with 24% acetonitrile in 0.1 M phosphate buffer (pH 4.75). The optimum electrochemical oxidation of EGF was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to electroactive tyrosine, tryptophan, methionine, and arginine residues. The quantitation range was from 1.0 to 200 ng of rhEGF with the linear correlation coefficient greater than 0.999. The method was successfully applied for the quantitation of rhEGF in a pharmaceutical preparation.

• 한국산림과학회지
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• 제80권4호
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• pp.436-444
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• 1991

이 연구(硏究)는 참나무류(類) 목재(木材)(수피(樹皮)포함)를 핀칩(pinchip)과 톱밥으로 만들어 이용(利用)하므로 목재 이용율(利用率)을 80%이상으로 활용할 수 있으며 이것에 표고버섯균의 활성(活性)을 조장(助長)시켜 버섯을 보다 효율적(效率的)으로 증수(增收)하고 가축(家畜)의 조사료(粗飼料)로 버섯재배 폐잔사(廢殘査)를 볏짚과 대체가능성(代替可能性)을 조사(調査) 연구(硏究)한 것이다. 그 결과(結果)는 다음과 같다. 1. 참나무류 칩을 이용한 버섯재배에서 버섯의 품질(品質)을 높이는 방법(方法)으로 유기산(有機酸)(탄닌산, 구연산)을 배양기(培養基)에 첨가(添加)하는 것은 균사발율(菌絲發育)과 품질(品質)에 큰 차이가 없는 것으로 나타났다. 2. 참나무류 칩이나 톱밥 재배는 종균(種菌) 접종(接種) 후(後) 6개월부터 제1차기 버섯수확(收穫)이 가능하면서 품질(品質)에서도 각처리구간(各處理區間)에 차이가 없는 것으로 나타났다. 3. 목재(木材)의 조단백질 함양은 0.7%인데 버섯재배 잔사는 1.82-4.55%까지 향상되었으며 총가소화(總可消化) 영양소율(營養素率)도 버섯재배잔사는 44.0-46.0%로 향상되어 볏짚 48.0%와 유의차(有意差)가 없었다. 4. 잔사중에 함유된 필수아미노산 함량을 조사한 바, 탄닌산처리 핀칩 및 톱밥잔사는 methionine이 그리고 무처리 핀칩 및 톱밥잔사의 경우는 isoleucine, phenylalanine, lysine 등이 비교구에 비하여 증가하였다. 반면에 threonine, valine, arginine 등은 비교구에 비하여 감소 추세였으며 특히 폐골목에서는 더욱 심한 현상을 보여주었다.

• 한국식품과학회지
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• 제28권4호
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• pp.684-688
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• 1996

전통식품인 된장으로부터 혈소판 응집 저해활성을 갖는 항혈전 펩타이드를 탐색하고자 하였다. 증류수로 추출한 된장액을 한외 여과하여 얻은 분자량 3,000 dalton 이하의 여액에 대하여, SD흰쥐의 세척혈소판 (washed platelet)을 이용하여 ADP 자극으로 유도되는 혈소판응집의 저해정도를 aggregometer에서의 탁도로 측정하였을 때, $96\;{\mu}g/ml$로 처리시 90% 가량의 응집 저해효과가 확인되었다. 추출물을 Dowex 50W X-2 강양이온 교환칼럼을 이용하여 pyridine-formic acid 완충액의 pH (2.8-5.0) 및 몰농도(0.1-3.0M)를 단계별로 증가시키면서 용출시켜 19개의 획분(No.B-18)으로 분리하였다. 이들 획분의 활성을 microplate를 이용한 방법으로 측정하였을 때, 전체적으로 앞의 방법보다는 감도가 낮게 나타났으나 모든 분획에서 응집억제 효과가 확인되었다. 이때 용량반응 곡선으로부터 구한 각 획분의 $IC_{50}$은 $10-1,000\;{\mu}g/ml)$의 범위를 보였으며, 대부분의 획분들은 양성대조구인 RGDS $(IC_{50},\;205\;{\mu}g/ml)$보다 높은 활성을 나타내었다. 또한, 후반부의 염기성 획분$(IC_{50},\;10-20\;{\mu}g/ml)$에서 된장추출물$(IC_{50},\;30\;{\mu}g/ml)$보다 활성이 높게 나타났다. 이들 중 활성이 가장 높은 16번 획분을 아미노산 분석한 결과, 펩타이드 부분에 histidine, arginine, alanine 등이 높은 비율로 존재하였다.

• Applied Biological Chemistry
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• 제40권3호
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• pp.178-183
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• 1997

한국재래간장으로 부터 분리한 Bacilius subtilis CCKS-111이 생성하는 proteae를 분리 정제하였다. 황산암모늄 염석, DEAE cellulose ion-exchange chromatography, Sephades G-100 및 HPLC를 이용한 겔여과법에 의해 비활성도 24.3 unit/mg, 정제배수 50.6배로 효소를 정제하였으며 high performance liquid chromatography (HPLC)에 의하여 단일 단백질임을 확인하였다 정제효소의 분자량은 HPLC에 의하여 분자량 28,000 정도의 monomer로 추정되었고, 본 효소의 아미노산 잔기수는 251.3으로, 분해되기 쉬운 threonine, serine, glycine을 제외한 아미노산 잔기수는 Bacillus subtilis subtilisin DY 잔기수(274)와 유사한 것으로 나타났다. 아미노산 조성은 alanine, serine, glycine 및 arginine의 함량이 많았다. Reverse phase (RP)-HPLC로 분리한 주 peak로 N-말단에서 32번 까지 아미노산 배열을 확인한 결과 Bacillus subtilis subtilisin DY와 동일하였다.