• Parasites, Hosts and Diseases
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• 제35권1호
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• pp.39-46
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• 1997

이 연구에서는 살아있는 질편모충의 단백질녈해효소가 인체 면역글로불린을 분해하는지 알아보고 질편모충에 의한 조직세포 독성에 있어서 단백질분해효소의 역할을 시험관내에서 관찰하였다 실험에 사용한 질편모충은 질염환자로년터 분리한 KT9 분리주이었으며 세포독성을 알아보기 위한 표적세포로는 HeLa 세포를 사용하였다 질편모충 단백질분해효소가 인체 면역글로불린을 분해하는지 관찰하고자 인체의 분비 IP. 혈청 IgA 및 IgG를 살아있는 원충. 원충의 용출액 및 분비-배설 액과 DTT를 넣어 반응시켰다. 여러 계열의 단백질분해효소 저해제(aminopeptidase, serine, metallo, cystelrle계열)를 살아있는 질편모충과 미리 반응시킨 후 세척하고 면역글로불린의 분해 단백질분해효소 활성 및 조직세포독성에 미치는 영향을 관찰하였다 살아있는 질편모충은 인체의 분비 IgA. 혈청 IgA 및 IgG를 분해하였는데 질편모충 수가 증가할수록 반응시간이 길수록 분해가 더 잘 이루어졌다 질푄모충의 용출액과 분비-배설액도 분비 IgA. 혈청 IgA 및 IgG를 분해하였다. Cysteine, serine계 열의 단백질분해효소 저해제 (I-64 antipain, iodoacetic acid, iodoacetamide, TLCK)를 처리한 질편닐충은 분비 IgA의 분해를 저해하였으며. 단백질분해효소저해제로 처리한 경우 질편모충의 단백질븐해효소 활성은 감소하였고 HeLa세포에 대한 독성이 감소하였다. 이상의 성적을 종합하면 질편모충에서 분비되는 단백질분해효소는 시험관내에서 조직세포에 세포독성을 나타내며 또한 인체면역글로불린을 분해하여 숙주의 방어기전에 대한 도피물질로 작용하는 것으로 생각된다.

• 농업과학연구
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• 제36권1호
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• pp.111-122
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• 2009

부들 화분(포항(蒲黃))의 혈전 용해능을 검토하기 위하여 부들 화분을 물 추출하여 혈전 분해능이 있음을 확인하였다. 부들 화분의 혈전용해효소를 DEAE-cellulose, Sephadex G-150을 이용한 gel filteration, HPLC로 정제하여 acrylamide gel electrophoresi로 정제를 하였다. 정제효소는 HPLC와 전기영동에 의하여 순수하게 정제되었음을 확인하였고 비활성은 38U/mg로서 정제도는 86.4배이었고 분자량은 75kDa이었다. 정제효소의 최적 pH는 4.0이었고 pH 4.0-6.0에서 안정하였으며 정제효소의 최적온도는 $55^{\circ}C$이었고 $30-60^{\circ}C$에서는 안정하였으나 $70^{\circ}C$ 부터 활성이 현저히 저하하여 $80^{\circ}C$ 이상에서는 완전히 실활하였다. 기질특이성은 fibrin을 가장 잘 분해하였고 fibrin, whole casein, ${\kappa}$-casein, ${\alpha}$-casein, ${\beta}$-casein, BSA순으로 나타났다. Casein을 기질로 하였을 때 Km value는 0.44 mM 이었으며 casein에 대한 친화력이 높은 것으로 나타났다. 효소활성에 미치는 금속이온의 영향은 $K^+$, $Na^+$, $Mg^{2+}$ 등은 효소반응에 영향을 미치지 않았으나 $Zn^{2+}$, $Fe^{2+}$는 심하게 저해작용을 나타내었고 정제효소는 PMSF, iodoacetic acid 및 SDS에 의하여 심하게 저해되는 것으로 보아 serine protease로 추정되었다.

• The Korean Journal of Physiology and Pharmacology
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• 제13권1호
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• pp.15-22
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• 2009

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide(KCN)/0.1 mM iodoacetic acid(IAA), and membrane transport function and cell viability were evaluated by measuring $Na^+$-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in $Na^+$-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited $Na^+$-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a $Na^+$ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in $Na^+$-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers(dimethylthiourea and thiourea), and amino acids(glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase $A_2(cPLA_2)$. The ATP depletion-dependent arachidonic acid release was inhibited by $cPLA_2$ specific inhibitor $AACOCF_3$. ATP depletion-induced alterations in $Na^+$-dependent phosphate uptake and cell viability were prevented by $AACOCF_3$. Inhibition of $Na^+$-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and $cPLA_2$ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

• Parasites, Hosts and Diseases
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• 제38권2호
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• pp.95-97
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• 2000

A 17 kDa protein from Clonorchis sinensis adults was purified by a procedure including Sephacryl S-200 HR gel filtration and Q-Sepharose anion exchange chromatography. The protein was proved to be a cysteine protease as it showed hydrolytic activity toward Cbz-Phe-Arg-AMC in the presence of dithiothreitol and was inhibited by specific inhibitors such as iodoacetic acid or trans epoxy-succinly-L-leucyl-amido(4 guanidino) butane. The polyclonal antibody raised against the protein reacted to 17 kDa proteins of trematodes such as Paragonimuf westermani, Fasciola hepatica, Opisthorchis viverrini, Gymnophalloides seoi, and Metagonimus yokogawai. The antibody recognized the 17 kDa and 16 kDa cysteine proteases purified from C. sinensis, P. westemani, and G. seoi as well. These results suggest that the 17 kDa protein may be a cysteine protease commonly present in trematodes.

• Archives of Pharmacal Research
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• 제27권8호
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• pp.878-883
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• 2004

Tacrolimus (FK506), which is isolated from Streptomyces tsukubaensis, is a new potent immu-nosuppressant. Because of poor solubility in water, the conventional intravenous dosage forms of tacrolimus contain surfactants such as cremophor EL (BASF Wyandotte Co.) or hydroge-nated polyoxy 60 castor oil (HCO-60) which may cause adverse effects. This study relates to a polymer-tacrolimus conjugate, which can be dissolved in water, formed by chemically binding the sparingly soluble drug, tacrolimus, with the water soluble polymer, methoxypoly(ethylene glycol) (mPEG). Water soluble tacrolimus-mPEG conjugates have been synthesized and shown to be function in vitro as prodrugs. These conjugates are in the form of an ester wherein the 24-, 32- or 24,32-positions are esterified. The desired 24-, 32- or 24,32-esterified com-pounds were obtained by initially acylating of tacrolimus with iodoacetic acid at the 24-,32-, or 24,32-positions and then reacting the resulting acylated tacrolimus with a mPEG in the pres-ence of a base such as sodium bicarbonate. These conjugates were converted again into tac-rolimus by the action of enzymes in human liver homogenate, and the half-lives of the conjugates are approximately 10 min in the homogenate, indicating that the esterified tacroli-mus derivatives may be practically applicable as a prod rug for the immunosuppressant.

• Archives of Pharmacal Research
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• 제24권5호
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• pp.446-454
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• 2001

Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at specific sites, were examined for the iodination of tyrosine residues. The 50 kDa polypeptide, which was prepared from digestion of bovine thyroglobulin and continuous preparative SDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-car-boxymethylated peptide derivative, which was further hydrohysed by endoproteinase-Asp-N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/ESI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase-Asp-N andthe mass spectra data, a peptide fragment turned out to correspond to a peptide, DALCCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another peptide fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyrosine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine residues, characteristic of a hormogenic site, existed in both peptides. In addition, the tyrosine residue in the peptide (1218-1252), corresponding to a hormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, it is suggested that polypeptide region adjacent to tyrosine residues may govern the susceptibility of tyrosine to oxidative iodination.

• Parasites, Hosts and Diseases
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• 제37권1호
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• pp.39-46
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• 1999

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection. a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HC1 (pH 7.4) containing 0.05. 0.1, 0.2 and 0.4 M NaC1 in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1. 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease. showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may playa role in the nutrient uptake of N. seoulense from the host intestine.

• Parasites, Hosts and Diseases
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• 제52권1호
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• pp.117-120
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• 2014

Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.

• 한방재활의학과학회지
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• 제30권2호
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• pp.1-18
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• 2020

Objectives This study intended to evaluate antioxidative, anti-inflammatory effects of Jibaekjihwang-tang on monosodium iodoacetate (MIA)-induced osteoarthritic rat model and investigate the potential mechanism. Methods Jibaekjihwang-tang (100 or 200 mg/kg body weight) was orally administered once daily for 2 weeks days from day 7 after intra-articular MIA injection. And blood analysis, the histologic examinations were performed. Moreover, protein expressions related to anti-oxidant and cartilage degradation and anti-inflammatory cytokines were measured by western blot analysis in cartilaginous tissue. Results Jibaekjihwang-tang reduced serum inflammatory cytokines such as tumor necosis factors-α and interleukin-6. Furthermore, the increase of anti-oxidant enzymes reversed the oxidative stress caused by MIA. Meanwhile, Jibaekjihwang-tang suppressed MIA-induced inflammation and cartilage degradation in cartilaginous tissue. Conclusions Jibaekjihwang-tang alleviated MIA-induced inflammation. Jibaekjihwang-tang was associated with a protective effect on cartilage and by reducing inflammation and cartilage degradation. These findings provide new approaches for understanding osteoarthritis therapy.

• 한국미생물·생명공학회지
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• 제17권6호
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• pp.545-551
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• 1989

알칼리성 Bacillus속 8-16 균주의 내열ㆍ알칼리성단백질 분해효소를 정제하여 그 특성을 조사하였다. 본 균주의 알칼리성 protease를 아세톤침전, CM-셀룰로즈크로마토그래피, Sephadex G-100 및 G-75 젤 여과법으로 정제하였고 비활성이 37배되게 하여 단일단백질이 될 때까지 순수정제하였다. 이 효소는 7$0^{\circ}C$ pH 11에서 pH 12 사이에서 최대 활성을 나타냈고 6$0^{\circ}C$에서는 한시간 동안 안정하였다. 이 효소의 $K_m$치는 1.3mg/$m\ell$이며 분자량 33,000으로 추정된다. 또한 이 효소는 Cu$^{2+}$ 및 Mn$^{2+}$에 의해서 약간 활성화되고 Ag$^+$, Hg$^{2+}$ 및 PMSF에 의해서 저해되므로 활성부위에 serine기가 관여하는 것으로 추정된다.