• Journal of Microbiology
• /
• v.33 no.3
• /
• pp.244-251
• /
• 1995

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.

• Journal of Photoscience
• /
• v.11 no.3
• /
• pp.133-139
• /
• 2004

The metal-ligand complex, $[Ru(phen)_2(dppz)]^{2+}$ (phen=1,10-phenanthroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), was used as a spectroscopic probe for studying nucleic acid dynamics. The RuPD complex displays a long lifetime and a molecular light switch property upon DNA binding due to shielding of its dppz ligand from water. To show the usefulness of this luminophore (RuPD) for probing nucleic acid dynamics, we compared its intensity and anisotropy decays when intercalated into the $tRNA^{val}$ and pBluescript (pBS) II SK(+) phagemid through a comparison with ethidium bromide (EB), a conventional nucleic acid probe. We used frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetime for the $tRNA^{val}$ (<${\tau}$> = 166.5 ns) was much shorter than that for the pBS II SK(+) phagemid (<${\tau}$> = 481.3 ns), suggesting a much more efficient shielding from water by the phagemid. Because of their size difference, the anisotropy decay data showed a much shorter rotational correlation times for the $tRNA^{val}$ (99.9 and 23.6 ns) than for the pBS II SK(+) phagemid (968.7 and 39.5 ns). These results indicate that RuPD can be useful for studying nucleic acid dynamics.

• 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.

• 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.

• The Korean Journal of Zoology
• /
• v.39 no.1
• /
• pp.36-46
• /
• 1996

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.

• Korean Journal of Food Science and Technology
• /
• v.36 no.3
• /
• pp.440-447
• /
• 2004

Characteristics of human ${\beta}-carotene$ 15,15'-dioxygenase isolated by recombinant DNA technology was elucidated. Optimal pH and temperature were 9.0 and $40^{\circ}C$, respectively. Enzyme activity was temperature-sensitive. Enzyme was stable at pH 6.0-9.0 for 24 hr and under $5^{\circ}C$. Half-life of enzyme at $35^{\circ}C$ was 40 min. Crude preparations of enzyme were inhibited by ferrous ion-chelating agent and sulfhydryl-binding agent. GSH offsets inhibitory effect of PCMB. With increasing substrate concentrations, enzyme activity gave typical Michaelis-Menten curve, Based on Hanes-Woolf plot of data, $K_{m}\;and\;V_{max$ were $3.39{\times}10^{6}\;M\;and\;1.2\;pmol/mg$ protein/min, respectively.

• Korean Journal of Materials Research
• /
• v.26 no.3
• /
• pp.117-122
• /
• 2016

White organic light-emitting diodes with a structure of indium-tin-oxide [ITO]/N,N-diphenyl-N,N-bis-[4-(phenylm-tolvlamino)-phenyl]-biphenyl-4,4-diamine [DNTPD]/[2,3-f:2, 2-h]quinoxaline-2,3,6,7,10,11-hexacarbonitrile [HATCN]/1,1-bis(di-4-poly-aminophenyl) cyclo -hexane [TAPC]/emission layers doped with three color dopants/4,7-diphenyl-1,10-phenanthroline [Bphen]/$Cs_2CO_3$/Al were fabricated and evaluated. In the emission layer [EML], N,N-dicarbazolyl-3,5-benzene [mCP] was used as a single host and bis(2-phenyl quinolinato)-acetylacetonate iridium(III) [Ir(pq)2acac]/fac-tris(2-phenylpyridinato) iridium(III) $[Ir(ppy)_3]$/iridium(III) bis[(4,6-di-fluoropheny)-pyridinato-N,C2] picolinate [FIrpic] were used as red/green/blue dopants, respectively. The fabricated devices were divided into five types (D1, D2, D3, D4, D5) according to the structure of the emission layer. The electroluminescence spectra showed three peak emissions at the wavelengths of blue (472~473 nm), green (495~500 nm), and red (589~595 nm). Among the fabricated devices, the device of D1 doped in a mixed fashion with a single emission layer showed the highest values of luminance and quantum efficiency at the given voltage. However, the emission color of D1 was not pure white but orange, with Commission Internationale de L'Eclairage [CIE] coordinates of (x = 0.41~0.45, y = 0.41) depending on the applied voltages. On the other hand, device D5, with a double emission layer of $mCP:[Ir(pq)_2acac(3%)+Ir(ppy)_3(0.5%)]$/mCP:[FIrpic(10%)], showed a nearly pure white color with CIE coordinates of (x = 0.34~0.35, y = 0.35~0.37) under applied voltage in the range of 6~10 V. The luminance and quantum efficiency of D5 were $17,160cd/m^2$ and 3.8% at 10 V, respectively.

• The Korean Journal of Mycology
• /
• v.22 no.2
• /
• pp.122-129
• /
• 1994

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.

• 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.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.7
• /
• pp.2117-2124
• /
• 2013

The binding properties and sequence selectivities of ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ (bip = 4,4'-biphenylene (imidazo [4,4-f][1,10]phenanthroline) complexes with $poly[d(A-T)_2]$ and $poly[d(G-C)_2]$ were investigated using conventional spectroscopic methods. When bound to $poly[d(A-T)_2]$, a large positive circular dichroism (CD) spectrum was induced in absorption region of the bridging moiety for both the ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complexes, which suggested that the bridging moiety sits in the minor groove of the polynucleotide. As luminescence intensity increased, decay times became longer and complexes were well-protected from the negatively charged iodide quencher compared to that in the absence of $poly[d(A-T)_2]$. These luminescence measurements indicated that Ru(II) enantiomers were in a less polar environment compared to that in water and supported by minor groove binding. An angle of $45^{\circ}$ between the molecular plane of the bridging moiety of the ${\Delta}{\Delta}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complex and the local DNA helix axis calculated from reduced linear dichroism ($LD^r$) spectrum further supported the minor groove binding mode. In the case of ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complex, this angle was $55^{\circ}$, suggesting a tilt of DNA stem near the binding site and bridging moiety sit in the minor groove of the $poly[d(A-T)_2]$. In contrast, neither ${\Delta}{\Delta}$-nor ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complex produced significant CD or $LD^r$ signal in the absorption region of the bridging moiety. Luminescence measurements revealed that both the ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complexes were partially accessible to the $I^-$ quencher. Furthermore, decay times became shorter when bis-Ru(II) complexes bound to $poly[d(G-C)_2]$. These observations suggest that both the ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ complexes bind at the surface of $poly[d(G-C)_2]$, probably electrostatically to phosphate group. The results indicate that ${\Delta}{\Delta}$- and ${\Lambda}{\Lambda}-[{\mu}-Ru_2(phen)_4(bip)]^{4+}$ are able to discriminate between AT and GC base pairs.