• YAKHAK HOEJI
• /
• v.40 no.6
• /
• pp.734-738
• /
• 1996

The development of the alpha2 isoform of (Na,K)ATPase which is high affinity ouabain receptors was studied in the differentiating nonfusing muscle cell line BC3H-1. T he differentiation process of BC3H-1 cell line was confirmed by 2-dexy-D-[$^3$H] glucose uptake experiment and the quantity of the expression of ${\alpha}_2$ isoform was measured using a whole cell [$^3$H] ouabain-binding assay. Undifferentiated growing BC3H-1 cells, myoblasts, exhibited low levels of insulin-stimulated glucose uptake and [$^3$H] ouabain-binding sites. In contrast, differentiated BC3H-1 cells, myocytes, had a 5.6-fold increase in insulin-stimulated glucose uptake and 5-fold increase in [$^3$H] ouabain-binding sites. Scatchard analysis showed that myocytes developed more [$^3$H] ouabain-binding sites than myoblasts vath a dissociation constant (kd) of 6${\times}10^{-8}$M and capacity of 6.l${\times}10^{-5}$ sites/cell. Therefore. it seems that myoblasts express low levels of ${\alpha}_2$ subunit and probably the majority of ${\alpha}_1$ subunit, whereas myocytes express high levels of ${\alpha}_2$ isoform. The results indicate that the expression of ${\alpha}_2$ isoform is developmentally regulated during differentiation and that BC3H-1 culture system provides an excellent model for the study of differentiation and mechanism of (Na,K)ATPase action in muscle which requires electrical excitability.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.30 no.2
• /
• pp.314-319
• /
• 2001

In the present study, effects of $\beta$-alanine, a known taurine antagonist for its structural similarity, on the adaptive regulation and kinetic behavior of the taurine transporter were investigated in the HT-29, human colon carcinoma cell line. Pretreatment of the cell with $\beta$-alanine(10mM) for varying periods from 3 to 30 hrs significantly reduced the taurine uptake compared to the value for control cells. This decrease in the taurine transporter activity was dependent on the incubation time with $\beta$-alanine, and the maximal down-regulation of the transporter activity was observed in cells pretreated with $\beta$-alanine for 24 hrs (25% of the control value, p<0.01). The taurine transporter appears to bind exclusively with $\beta$-alanine in the HT-29 cells since the same concentration of $\alpha$-alanine added in the culture medium for 24 hrs did not influence the taurine uptake. Kinetic analyses of the taurine transporter activity was performed in the HT-29 cell line with varying taurine concentration (5~60$\mu$M) in the uptake medium. Active taurine uptake was significantly lower in $\beta$-alanine pretreated cells compared to the value for control cells in the range of taurine concentration used in the experiment (p<0.001). The cells pretreated with $\beta$-alanine showed a 50% lower maximal velocity (Vmax, 1.7$\pm$2.0 nmole.mg $protein^{-1}$.$30min^{-1}$), and a 99% higher Michaelis constant (Km, 40.3$\pm$7.6$\mu$M) than the control values (3.3$\pm$1.9 nmole.mg $protein^{-1}$.$30min^{-1}$, and 20.3$\pm$2.1$\mu$M, respectively). These results on kinetic data suggest that $\beta$-alanine induced down-regulation of the taurine transporter activity was associated with decreases in both maximal velocity and affinity of the transporter.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.12
• /
• pp.2087-2097
• /
• 2016

Most cold-adapted enzymes possess higher $K_m$ and $k_{cat}$ values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near $K_m$. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately $30^{\circ}C$ and $40^{\circ}C$, respectively. The melting points were $33^{\circ}C$ and $45^{\circ}C$ for CpSK and EcSK, respectively. The ${\Delta}G_{H_2O}$ (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower $K_m$ for its substrate shikimate than most mesophilic SKs, and the $k_{cat}$ was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

• Food Science and Biotechnology
• /
• v.14 no.6
• /
• pp.727-731
• /
• 2005

Synthesized Ile-Ala-Val-Pro (IAVP) peptide, which has the highest hypocholesterolemic effect among a number of synthesized derivatives of Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA) isolated from 11S globulin of soy protein by pepsin digestion, was selected for investigation in the present study. Using a recombinant Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), we studied in detail the inhibition of this enzyme by IAVP and compared the action of this peptide to that of lovastatin, a known competitive inhibitor of this enzyme. The concentration of IAVP required for 50% inhibition ($IC_{50}$) of HMGR activity in given experimental conditions was $340\;{\mu}M$. Kinetic analysis revealed that the studied peptide is a competitive inhibitor of HMGR with respect to both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) and nicotinamide adenine dinucleotide phosphate (NADPH), with an equilibrium constant of inhibitor binding ($K_i\;=\;[E][I]/[EI]$) of $61{\pm}1.2\;{\mu}M$ and $157{\pm}4.4\;{\mu}M$, respectively. At the same conditions, $K_i$ and $IC_{50}$ for lovastatin were $2.2{\pm}0.1\;nM$ and 12.5 nM, respectively. Thus, the given peptide interacts with HMGR as a bisubstrate, consequently blocking access of both substrates to the active sites. The achieved results suggest the design of new peptide sequences having a higher relative affinity to binding sites of this enzyme and an enhancement of their hypocholesterolemic properties.

• Journal of the Korean Society of Tobacco Science
• /
• v.29 no.2
• /
• pp.98-103
• /
• 2007

This study compares menthol migration from fillers to filters and mainstream smoke in a type of leaf tobacco and according to the moisture content differences at the range of $11{\sim}15\;%$. The leaf tobacco used in this study consisted of Korea flue cured upper leaves B1O (KFUB1), A40R (KFUA4), lower leaves C1L (KFLC1), CD4L (KFLCD4), burley upper leaves A3T (KBUA3), lower leaves D3W (KBLD3), Orient Basma I/III (OB), Orient Izmir BIG (OI), expanded tobacco (KET), and reconstituted sheet (KRC). Menthol migration to the filter and mainstream was measured under constant conditions for 80 days with intervals of 20 days. In the comparison between flue cured types, there were significant differences in the filter parts, as follows. KFUB1(34.4 %) KFUA4(37.4 %), KFLC1(43 %) and KFLCD4 were 55.7 %. In the comparision between other types of leaf tobaccos, KFUB1 was 34.4 % and KET was 52.6 % at filter parts. In the methol transfer to mainstream smoke was $16.5{\sim}24.2\;%$. The menthol migration to filters was measured based on the moisture content of $11{\sim}15\;%$ for the KFUB1 after storing it for 80 days. The menthol migrations were $36{\sim}40\;%$ at the moisture content of $11{\sim}15\;%$, respectively. The transfers to mainstream smoke were $12.8{\sim}15.8\;%$.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1995.04a
• /
• pp.81-81
• /
• 1995

It is well known that Src Homology 2(SH2) domain in many intracellular signal transduction proteins is very important. The domain has about 100 amino acid residues and bind phosphotyrosine-containing peptide with high affinity and specificity. Lck SH2 domain is a Src-like, lymphocyte-specific tyrosine kinase. An 11-residue phosphopeptide derived from the hamster polvoma middle-T antigen, EPQp YEEIPIYL, binds with an 1 nM dissociation constant to Lck SH2 domain. And it is known that the phosphotyrosine and isoleucine residues of the peptide are tightly bound by two well-defined pockets on Lck SH2 domain's surface. To investigate the conformational changes during complexation of SH2 domain with phosphopeptide we have performed the molecular dynamics simulation for Lck SH2 domain with peptide and peptide-free form at look in aqueous solution. More than 3000 water molecules were incorporated to solvate Lck SH2 domain and peptide. Periodic boundary condition has been applied in molecular dynamics simulation. Data analysis with the results of that simulation shows that the phosphopeptide makes primary interaction with the Lck SH2 domain at six central residues, The comparison of the complexed and uncomplexed SH2 domain structures in solution has revealed only relatively small change. But the hydrophilic and hydrophobic pockets in the protein surface show the conformational changes in spite of the small structural difference between the complex and peptide-free forms.

• Korean Journal of Food Science and Technology
• /
• v.21 no.4
• /
• pp.569-574
• /
• 1989

These studies were conducted to investigate the purification and characterization of Kiwifruit protease, and the results obtained were as follows The protease was purified by ammonium sulfate fractionation, Sephadex G-100 filtration and DEAE-Sephadex A-50 column chromatography and purified enzyme gave a single protein band on polyacrylamide gel electrophoresis The specific activity of purified enzyme was 30,10 units/mg protein and the yield was 7.48. The purified enzyme showed a high affinity for casein and hemoglobin. The optimal pH and temperature for enzyme activity were 7.0 and $45^{\circ}C$, respectively. The enzyme activity was strongly inhibited by $HgCl_2,\;MnSO_4$. However. the enzyme was activated by cysteine and EDTA. The Michaelis constant for casein was calculated to be 50.5mg/ml according to the Line weaver-Burk method, and its molecular weight was determied as 23,500 by polyacrylamide gel electrophoresis.

• Microbiology and Biotechnology Letters
• /
• v.33 no.2
• /
• pp.106-111
• /
• 2005

To investigate the substrate variety of a putative non-metal dependent isomerase, the tagatose-6-phosphate isomerase (E.C. 5.3.1.26) structural genes (lacB; 510bp and lacA; 430bp) of Staphylococcus aureus were subcloned and co-expressed. Based on the substrate configuration, various aldoses were surveyed for substrate of ketose isomerization. Among the 10 aldoses tested, D-ribose and D-allose were isomerized by the enzyme. The subunit A and B showed more than $95\%$ activity for D-ribose and $75\%$ for D-allose in the presence of 1mM EDTA compared with non-EDTA conditions, which implying tagatose-6-phosphate isomerase is a non-metal dependent isomerase. Each of subunit A or subunit B alone showed no activity for any of the substrates tested. The affinity constant ($K_m$) of tagatose-6-phosphate isomerase against D-ribose and D-allose were 26 mM and 142 mM, respectively.

• Animal cells and systems
• /
• v.1 no.1
• /
• pp.77-80
• /
• 1997

DOantrolene is a primary specific therapeutic drug for prevention and treatment of malignant hyperthermia symptoms. The mechanisms underlying the therapeutic effects of the drug are not well understood. The present study aimed at the characterization of the effects of azumolene, a water soluble dantrolene analogue, on ryanodine binding to sarcoplasmic reticulum (SR) from normal and malign::lnt hyperthermia susceptible (MHS) swine muscles. Characteristics of $[^3H]ryanodine$ binding were clearly different between the two types of SR. Kinetic analysis of eH]ryanodine binding to SR in the presence of $2{\mu}M$ $Ca^{2+}$ showed that association constant $(K_{ryanodine}_7$ is significantly higher in MHS than normal muscle SR $(2.83 vs. 1.32{\times}10^7 M^{-1}$, whereas the maximal ryanodine binding capacity $(B_{max})$ is similar between the two types of SR. Addition of azumolene $(e.g. 400{\mu}M)$ did not significantly alter both $K_{ryanodine}$ and $B_{max}$ of $[^3H]$ryanodine binding in both types of SR, indicating that the azumolene effect was not on the ryanodine binding sites. Addition of caffeine activated $[^3H]$ ryanodine binding in both types of SR, and caffeine sensitivity was significantly higher in MHS muscle SR than normal muscle SR $(K_{caffeine}:3.24 vs. 0.82 {\times} 10^2 M^{-l}). Addition of azumolene $(e.g.400{\mu}M)$ decreased Kcaffeine without significant change in $B_{max}$ in both types of SR suggesting that azumolene competes with caffeine binding site(s). These results suggest that malignant hyperthermia symptoms are caused at least in part by greater sensitivity of the MHS muscle SR to the $Ca^{2+}$ release drug(s), and that azumolene can reverse the symptoms by reducing the drug affinity to $Ca^{2+}$ release channels.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.4
• /
• pp.1025-1029
• /
• 2013

In the previous paper (Bull. Korean Chem. Soc., 2011, 32, 2997), the hybrid molecules between ${\alpha}$-lipoic acid (ALA) and polyphenols (PPs) connected with neutral 2-(2-aminoethoxy)ethanol linker (linker-1) showed new biological activity such as butyrylcholinesterase (BuChE) inhibition. In order to increase the binding affinity of the hybrid compounds to cholinesterase (ChE), the neutral 2-(2-aminoethoxy)ethanol (linker 1) was switched to the cationic 2-(piperazin-1-yl)ethanol linker (linker 2). The $IC_{50}$ values of the linker-2 hybrid molecules for BuChE inhibition were lower than those of linker-1 hybrid molecules (except 9-2) and they also had the same great selectivity for BuChE over AChE (> 800 fold) as linker-1 hybrid molecules. ALA-acetyl caffeic acid (10-2, ALA-AcCA) was shown as an effective inhibitor of BuChE ($IC_{50}=0.44{\pm}0.24{\mu}M$). A kinetic study using 7-2 showed that it is the same mixed type inhibition as 7-1. Its inhibition constant (Ki) to BuChE is $4.3{\pm}0.09{\mu}M$.