Yong-Kweon Cho

In vivo effects of panax ginseng extracts on the cytochrome p450-dependent monooxygenase system in the liver of 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed guinea pig

The effects of the subchronic administration of Panax ginseng extracts were examined on the hepatic cytochrome P450-dependent monooxygenase system of guinea pigs pre-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Panax ginseng extracts were intraperitoneally administered to guinea pigs at 100 mg/kg/day for 14 days from 1 week after a single intraperitoneal injection of 1 microg of TCDD/kg of body weight. TCDD treatment increased the total cytochrome P450 content 2.86-fold, and this was remarkably inhibited by the administration of Panax ginseng extracts. Treatment with ginseng extract alone also decreased the contents of cytochrome P450 by 33%, but both TCDD and ginseng extracts had no effect on cytochrome b(5) content. The administration of TCDD resulted in a 1.73-fold increase in microsomal NADPH-cytochrome P450 reductase activity in the guinea pig liver, and this was significantly inhibited by ginseng extracts, but treatment with ginseng extracts alone had no effect on its activity, and no statistical changes in the activity of NADPH-cytochrome b(5) reductase were observed in guinea pig liver due to TCDD and/or ginseng extract administration. Compared to the control, ECOD activity remarkably (1.76-fold) increased after TCDD administration, but this increase was completely inhibited by treatment with ginseng extract. Treatment with ginseng extract alone resulted in a 50% reduction of ECOD activity. TCDD administration remarkably induced benzphetamine demethylation (BPDM) activity, while ginseng extract also slightly increased the enzymes activity, but the induction attributed to ginseng extracts was not statistically significant. Even though administration of ginseng extracts slightly inhibited TCDD-induced BPDM activity, the inhibition was not statistically significant. These results indicate that ginseng extract exerts different effect on the induction of P450 isozymes. From these results, we suggest that Panax ginseng extracts may act as an inhibitor of CYP1A rather than that of CYP2B.

Effects of pressure on the kinetics of capture by yeast alcohol dehydrogenase.

High pressure causes biphasic effects on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase as expressed in the kinetic parameter V/K which measures substrate capture. Moderate pressure increases the rate of capture of benzyl alcohol by activating the hydride transfer step. This means that the transition state for hydride transfer has a smaller volume than the free alcohol plus the capturing form of enzyme, with a ΔV⧧ of −39 ± 1 mL/mol, a value that is relatively large. This is the first physical property of an enzymatic transition state thus characterized, and it offers new possibilities for structure−activity analyses. Pressures of >1.5 kbar decrease the rate of capture of benzyl alcohol by favoring a conformation of the enzyme which binds nicotinamide adenine dinucleotide (NAD+) less tightly. This means that the ground state for tight binding, E*−NAD+, has a larger volume than the collision complex, E−NAD+, with a ΔV* of 73 ± 2 mL/mol. The equilibrium constant of the conformational change K...

Effects of High Pressure on Solvent Isotope Effects of Yeast Alcohol Dehydrogenase

The effect of pressure on the capture of a substrate alcohol by yeast alcohol dehydrogenase is biphasic. Solvent isotope effects accompany both phases and are expressed differently at different pressures. These differences allow the extraction of an inverse intrinsic kinetic solvent isotope effect of 1.1 (i.e., (D(2(O)))V/K = 0.9) accompanying hydride transfer and an inverse equilibrium solvent isotope effect of 2.6 (i.e., (D(2(O)))K(s) = 0.4) accompanying the binding of nucleotide, NAD(+). The value of the kinetic effect is consistent with a reactant-state E-NAD(+)-Zn-OH(2) having a fractionation factor of phi approximately 0.5 for the zinc-bound water in conjunction with a transition-state proton exiting a low-barrier hydrogen bond with a fractionation factor between 0.6 and 0.9. The value of the equilibrium effect is consistent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the binding of NAD(+). The absence of significant commitments to catalysis accompanying the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concerted chemical mechanism. The success of this analysis suggests that future measurements of solvent isotope effects as a function of pressure, in the presence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture at atmospheric pressure.

Characterization and Purification of the Bacteriocin Produced by Bacillus licheniformis Isolated from Soybean Sauce

A bacteriocin-producing bacterium identified as Bacillus licheniformis was isolated from soybean sauce. Antibacterial activity was confirmed by paper disc diffusion method, using Micrococcus luteus as a test organism. The bacteriocin also showed antibacterial activities against Bacillus sphaericus, Lactobacillus bulgaricus, Lactobacillus plantarum, Paenibacillus polymyxa, and Pediococcus dextrinicus. Optimal culture conditions for the production of bacteriocin was attained by growing the cells in an MRS medium at a pH of 6.5～7.0 and a temperature of 37℃ for 36～48 hr. Solvents such as chloroform, ethanol, acetone, and acetonitrile had little effect on bacteriocin activity. However, about 50% of bacteriocin activity diminished with treatment of methanol and isopropanol at the final concentration of 50% at 25℃ for 1 hr. It was stable against a pH variation range from 3.0 and 7.0, but the activity reduced to 50% at a pH range from 9.0 to 11.0. Its activity was not affected by heat treatment at 100oC for 30 min and 50% of activity was retained after heat treatment at 100℃ for 60 min, showing high thermostability. The bacteriocin was purified to a homogeneity through ammonium sulfate precipitation, SP-Sepharose ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC). The entire purification protocol led to a 75-fold increase in specific activity and a 13.5% yield of bacteriocin activity. The molecular weight of purified bacteriocin was estimated to be about 2.5 kDa by tricine-SDS-PAGE.Characterization and Purification of the Bacteriocin Produced by Bacillus licheniformis Isolated from Soybean Sauce. Sung-Sub Jung 1 , Jung-I Choi 1 , Woo-Hong Joo 2 , Hyun-Hyo Suh 3 , Ae-Sil Na, Yong-Kweon Cho, Ja-Young Moon, Kwon-Chul Ha, Do-Hyeon Paik and Dae-Ook Kang*. Department of Biochemistry and Health Science, Changwon National University, Changwon 641-773, Korea, Interdisciplinary Program in Biotechnology, Graduate School, Changwon National University, Department of Biology, Changwon National University, Department of Environmental Engineering, Jinju National University, Jinju 660-758, Korea A bacteriocin-producing bacterium identified as Bacillus licheniformis was isolated from soybean sauce. Antibacterial activity was confirmed by paper disc diffusion method, using Micrococcus luteus as a test organism. The bacteriocin also showed antibacterial activities against Bacillus sphaericus, Lactobacillus bulgaricus, Lactobacillus plantarum, Paenibacillus polymyxa, and Pediococcus dextrinicus. Optimal culture conditions for the production of bacteriocin was attained by growing the cells in an MRS medium at a pH of 6.5~7.0 and a temperature of 37 o C for 36~48 hr. Solvents such as chloroform, ethanol, acetone, and acetonitrile had little effect on bacteriocin activity. However, about 50% of bacteriocin activity diminished with treatment of methanol and isopropanol at the final concentration of 50% at 25 o C for 1 hr. It was stable against a pH variation range from 3.0 and 7.0, but the activity reduced to 50% at a pH range from 9.0 to 11.0. Its activity was not affected by heat treatment at 100 o C for 30 min and 50% of activity was retained after heat treatment at 100 o C for 60 min, showing high thermostability. The bacteriocin was purified to a homogeneity through ammonium sulfate precipitation, SP-Sepharose ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC). The entire purification protocol led to a 75-fold increase in specific activity and a 13.5% yield of bacteriocin activity. The molecular weight of purified bacteriocin was estimated to be about 2.5 kDa by tricine-SDS-PAGE.

Transpeptidation by Porcine Pepsin Catalyzed by a Noncovalent Intermediate Unique to Its Iso-mechanism

Porcine pepsin proteolysis of the hexapeptide Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe (where OMe = methoxy and Nle = norleucine) in the presence of dipeptide Leu-Leu synthesizes a new hexapeptide Leu-Ser-p-nitro-Phe-Leu-Leu. Contrary to transpeptidation kinetics of other proteases, which depend upon an acyl-enzyme intermediate, the time course for pepsin-catalyzed transpeptidation displays a distinct lag before reaching a steady-state reaction velocity. Moreover, this lag is coupled to burst kinetics for the formation of proteolytic products, Leu-Ser-p-nitro-Phe and Nle-Ala-Leu-OMe. The lag requires that free Leu-Ser-p-nitro-Phe accumulate in the reaction medium during the lag phase and subsequently rebind for transpeptidation. Consistent with this dissociative kinetic mechanism are normal solvent isotope effects on formation of the proteolytic products Leu-Ser-p-nitro-Phe (v H /v D = 2.2 ± 0.2) and Nle-Ala-Leu-OMe (v H /v D = 1.8 ± 0.1) as opposed to an inverse effect on the formation of the transpeptidation product Leu-Ser-p-nitro-Phe-Leu-Leu (v H /v D = 0.40 ± 0.09). Because proteolysis is slower in D2O but transpeptidation is faster, the isotopically sensitive step must occurafter release of both products of proteolysis, which precludes putative acyl-enzyme covalent intermediates. Isotopically enhanced transpeptidation is a new type of isotope effect but one that is consistent with the Uni Bi iso-mechanism previously postulated on the basis of solvent isotope effects on V maxbut not on V max/K m (Rebholz, K. L., and Northrop, D. B. (1991) Biochem. Biophys Res. Commun. 179, 65–69) and confirmed by solvent isotope effects on the onset of inhibition by pepstatin (Cho, Y.-K., Rebholz, K. L., and Northrop, D. B. (1994)Biochemistry 33, 9637–9642). As a new biochemical mechanism for peptide bond synthesis that has a potential for applications in biotechnology, it is here proposed that the energy necessary to drive peptide synthesis from free peptides comes from the sizable free energy drop associated with rehydration of the active site of pepsin in 55 m water.

The Signal Sequence of Sporulation-Specific Glucoamylase Directs the Secretion of Bacterial Endo-1,4-β-D-Glucanase in Yeast

The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4- -D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.