To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.
Park, Eun-Mi;Park, Ji-Sun;Kim, Yun-Jeong;Sung, Jae-Suk;Hwamg, Tea-Sook;Kim, Woo-Chul;Han, Mi-Young;Park, Young-Mee
BMB Reports
/
v.34
no.6
/
pp.544-550
/
2001
In pre-transplant total-body irradiation (TBI), the lung is a critical dose-limiting organ. Also, the possible role of oxidative stress was suggested in the development of TBI-induced lung damage. This study explores the association between TBI-induced oxidative stress and the induction of lung pathogenesis by investigating TBI-induced oxidative stress in the lungs of male C57BL/6 mice after a single dose of 10 Gy TBI. We showed significant increases of reactive oxygen species (ROS) formation and lipid peroxidation, and also a depletion and oxidation of glutathione after TBI. There is evidence that pretreatment with 1,10-phenanthroline (o-phen) significantly reduces oxidative stress in the lung. This indicates that the TBI-induced ROS generation involves a metal-catalyzed Fenton-type reaction. A pretreatment of buthionine sulfoximine (BSO) augmented the glutathione depletion and oxidation, but had no effect on the ROS formation and lipid peroxidation up to 6 h after TBI. Histopathological features that are consistent with pneumonitis were observed in the BSO pretreated-mice 1 week after irradiation. The results suggest that TBI-induced oxidative stress in the lung involves a generation of ROS through a Fenton-type reaction. Also, glutathione plays an important inhibitory role in the radiation-induced lung pathogenesis by participating in the self-amplifying cascade subsequent to the ROS generation by irradiation.
Kim, Joung-Yoon;Lee, Seung-Bae;Kwon, Ki Rok;Choi, Suk-Ho
Journal of Pharmacopuncture
/
v.17
no.1
/
pp.44-50
/
2014
Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.
A double-layer optical sensing membrane has been fabricated to measure the concentration of dissolved oxygen (DO) and pH value simultaneously. (tris(4,7-diphenyl-1,10-phenanthroline (Rudpp) ruthenium(II)) as a DO sensitive dye has been mixed in the methyl trimethoxy silane (MTMS) sol-gel solution and coated onto one well in a 24-well microtiter plate. On the DO-sensing layer the GA (3-glycidoxypropyl trimethoxy silane (GPTMS), 3-aminopropyl trimethoxy silane (APTMS)) sol-gel solution mixed with 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) has been coated and used to measure pH values. The double-layer sensing membrane was affected by ionic strength and temperature. The double-layer sensing membrane for DO and pH has been applied to online monitor in microorganism cultivation processes and showed a good performance.
An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca$\^$2+/, Zn$\^$2+/, Fe$\^$2+/, Mg$\^$2+/ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40.deg.C. It was stable at least for 1 week at 40.deg.C and maintained its activity for 24 hours at 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.
The role of metal ions for the activity of the mitochondrial $F_1-ATPase$ was studied. Removal of non-heme iron ion from the mitochondria by dialysis against chelating agents, 10 mM ethylenediaminetetraacetic acid(EDTA) and 10 mM o-phenanthroline(o-Phe), led to 56% and 49% inactivation of the enzyme, respectively. The enzyme dialyzed against EDTA was reactivated 81% by the addition of 0.5 mM $Fe^{3+}$ and 70% by 0.5 mM $Mg^{2+}$. But, $Fe^{2+}$ did not reactivate the enzyme. Coexistence of 0.5 mM $Fe^{2+}$ and 0.5 mM $Mg^{2+}$ resulted in 95% reactivation of the enzyme, while $Fe^{3+}$ with 0.5 mM $Mg^{2+}$ did not reactivate the enzyme like the effect of $Fe^{2+}$ alone. The enzyme dialyzed against o-Phe showed the similar results. These data showed that $Fe^{3+}$ is predominantly required for the activity of the mitochondrial $F_1-ATPase$ in Lentinus edodes and stimulated the activity of it by $Mg^{2+}$. $Fe^{3+}$ and $Mg^{2+}$ increased enzyme's affinity for substrate, decreasing the Km value 1.67 mM to 0.65 mM.
An endogenous phenoloxidase (EPO) from earthworm, Lumbricus rubellus, has been purified and characterized. The purified EPO using ammonium sulfate fractionation, Blue-2, Phenyl-, and Q-sepharose chromatography steps was revealed in SDS-PAGE as a single protein banri with Mr. of 59 kl)a. A native strudure of the enzyme was examined with an in situ staining of a nondenatudng-PAGE using DL-dopa as a substrate. The result showed that a single band due to the EPO activity was located siighdy above a standard polypeptide with Mr. of 210 kl)a. These fads indicate that the EPO is an oligomeric enzyme. The presence of a monophenolase activity of the purified EPO, which hydroxylates tyrosine to dopa, was confirmed by observing dopachrome accumulation at 475 nm at PH 8.0 with a typical lag phase during 60 mm. of meausrement. A series of inhibition study has been performed for the enzyme with several divalent cation chelators such as phenyithiourea (Flu), 1, lO-phenanthroline, EDTA, and EGTA. Among them, only V'flj inhibited the enzyme with 1C0.5 of 65 MM, which indicated that copper was critical for the catalysis of EPO. The enzyme was maximally active at 35'C and pH 8.0 when L-dopa to dopachrome conversion was spectrophotometricaily monitored at 475 nm. The apparent Km values of P0 for L-opa were obtained as 1.86 mM and 13.8 mM at pH 6.5 and 8.0, respectively. The catalytic efficiencies at both pH were almost identical [(kat/Km)pH8.0/(kcat/Km)pH6.5 = O.92] while the Vmax at p11 8.0 was 6.6-fold higher than that at pH 6.5. This fact may indicate that pH affeds the catalysis at substrate and/or enzyme-substrate complex level rather than the enzyme itself. Taken together, the EPO was an oligomeric enzyme which did not require proteolysis for its activation. These results also indicated that the enzyme can exist, at least, in part as a latent form In vivo, which might be distinct from the prophenoloxidase activating system. Therefore, it is pertinent to consider that there must be certain regulatory molecules or phenomena in L. rubellus which make the 1,0 in a latent form in vivo before the foreign invasions.
Mansouri-Torshizi, Hassan;Saeidifar, Maryam;Ghasemi, Zahra Yekke;Khastan, Mahmood;Divsalar, Adeleh;Saboury, Ali Akbar
Journal of the Korean Chemical Society
/
v.55
no.1
/
pp.70-80
/
2011
Two new palladium (II) complexes, [Pd (phen)(pip-dtc)]$NO_3$ and [Pd(phen)(mor-dtc)]$NO_3$, (where phen is 1,10-Phenantroline, pip-dtc is piperidinedithiocarbamate anion and mor-dtc is morpholinedithiocarbamate anion) have been synthesized and characterized by elemental analysis, spectroscopic studies (FT-IR, $^1H$ NMR, UV-Vis) and conductance measurement. In these complexes, the dithiocarbamate ligands coordinate with Pd (II) center as bidentate with two sulfur atoms. These two complexes have been tested against chronic myelogenous leukemia cell line, K562. They show $IC_{50}$ values less than cisplatin and thus the mode of binding of the complexes to calf thymus DNA (CT-DNA) were investigated by ultraviolet difference and fluorescence spectroscopy. They can denature DNA, exhibit cooperative binding and intercalate into DNA. Several binding and thermodynamic parameters are also described.
Kim, Min-Jung;Kim, Ji-Young;Leec, Seung-Jae;Yoon, Yong-Dal;Cho, Dong-Jae;Kim, Hae-Kwon
Development and Reproduction
/
v.5
no.1
/
pp.23-33
/
2001
When mammalian oocytes ovulate into the oviduct, associating follicular fluid components are exposed to the oviductal environment, possibly resulting in the mutual interaction between fillicu1ar and oviductal fluids. In the Present study, we have demonstrated for the first time that components of fallicular fluid could be modified by the oviductal fluid. Gelatin zymographic analyses of human follicular fluid (hFF) obtained from IVF patients showed consistently the presence of 110 kDa gelatinase (GA110) in addition to many bands among which 62 kDa gelatinase was predominant. Addition of EDTA or phenanfhroline to the gelatinase substrate buffer during gel incubation abolished GA110 band whereas phenylmethylsulffnyl fluoride (PMSF) did not. In contrast, bovine oviductal fluid(bOF) exhibited only 62 kDa gelatinase. Surprisingly, when bOF was added to hFF in 1:1 ratio and then the mixture was incubated for 3 h at 37$^{\circ}$C, GA110 of hFF disappeared. Disappearance of GA110 by bOF was observed even within 30 min after mixing with hFF. Addition of aminophenylmercuric acetate (APMA) to hFF also abolished enzymatic activity of GA110 but increased the activityof 62 kDa gelatinase. However, APMA abolished many other gelatinases as well unlike bOF. Interestingly, treatment of hFF with EDTA for 3 h remarkably increased the enzymatic activity of GA110 but not that of other gelatinases. Addition of phenanthroline, PMSF or soybean trypsin inhibitor (SBTI) did not affect overall gelatinase activities. Again, addition of bOF to the hFF pretreated with any of the above proteinase inhibitors abolished the appearance of GA110. Human serum also showed GAI 10 of which activity was greatlyenhanced by EDTA treatment. Similar to hFF, serum GA110 also disappeared by the addition of bOF. Human granulosa cell homogenate did not reveal any appreciable gelatinase activity except 92 kDa gelatinase. Anti-human gelatinase A antibody reacted with 62 kDa gelatinase of hFF. Based upon these results, it is concluded that bOF could selectively degrade an isoform of gelatinase A present in hFF and human serum.
SOHN Heung-Sik;PARK Seong-Min;SON Byung-Yil;CHOI Hyeon-Mee;LEE Keun-Tai
Korean Journal of Fisheries and Aquatic Sciences
/
v.32
no.2
/
pp.121-126
/
1999
In order to degrade chitin by enzymatic hydrolysis, it is required from screening highly active deacetylase. To this end, we examined three fungal strains and it turned out that Mucor rouxii produced highly active deacetylase, this enzyme exhibited the highest enzymatic activity against colloidal chitin. The conditions for growing Mucor rouxii are as follows; the effective carbon source, nitrogen source, adequate initial pH, temperature and incubation time were $2\%$ glucose, $1.33\%$ yeast extract, $0.66\%$ pepton, 4.5, $25{\pm}2^{\circ}C$ and 48hr, respectively. The optimum pH and temperature for purified enzyme activity were 5.5 and $40^{\circ}C$, respectively. The purified enzyme was stable at pH ranging from 4.5 to 5.5. However, the enzyme activity was decreased to less than $50\%$ at pH blow 45 and above 7.5. At temperatures above $50^{\circ}C$, the enzyme activity was decreased remarkably. The enzyme was inhibited by LiC1, $HgCl_2$, and $BaCl_2$, but stimulated by $CaCl_2$ and $ZnC1_2$, The activity of purified enzyme was increased by L-cysteine and 2-mercaptoethanol, while decreased by O-phenanthroline, p-CMB, EDTA, and iodoacetate. The $K_m$ and the $V_{max}$ value of purified enzyme were $1.2\%$ and 59.5 U/mg, respectively. The deacetylation activity of purified enzyme was not detected at optimal reaction condition when chitin particle suspension was used.
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