• BMB Reports
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• v.37 no.5
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• pp.515-521
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• 2004

The tricarboxylate (or citrate) carrier was purified from eel liver mitochondria and functionally reconstituted into liposomes. Incubation of the proteoliposomes with various sulfhydryl reagents led to inhibition of the reconstituted citrate transport activity. Preincubation of the proteoliposomes with reversible SH reagents, such as mercurials and methanethiosulfonates, protected the eel liver tricarboxylate carrier against inactivation by the irreversible reagent N-(1-pyrenyl)maleimide (PM). Citrate and L-malate, two substrates of the tricarboxylate carrier, protected the protein against inactivation by sulfhydryl reagents and decreased the fluorescent PM bound to the purified protein. These results suggest that the eel liver tricarboxylate carrier requires a single population of free cysteine(s) in order to manifest catalytic activity. The reactive cysteine(s) is most probably located at or near the substrate binding site of the carrier protein.

• The Korean Journal of Physiology
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• v.25 no.2
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• pp.179-188
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• 1991

Effect of a sulfhydryl reagent, p-chloromercuribenzoic acid (PCMB), on the transport of succinate was studied in brush border (BBMV) and basolateral (BLMV) membrane vesicles isolated from rabbit renal cortex. PCMB induced an irreversible inhibition of the $Na^+-dependent$ succinate uptake in a dose-dependent manner with $IC_{50}$ of 55 and $65\;{\mu}M$ in BBMV and BLMV, respectively. The inhibitory effect of PCMB was prevented by a pretreatment of vesicles with dithiothreitol. PCMB did not increase $Na^+$ permeability at concentrations inhibiting succinate uptake. The PCMB inhibition of succinate uptake was due to a change in Vmax, but not in Km. When membrane vesicles were pretreated with PCMB in the presence of unlabelled succinate, the inhibitory effect was significantly reduced. In both BBMV and BLMV, succinate uptake was inhibited by various sulfhydryl reagents with the inhibitory potency of following order: $HgCl_2$>DTNB>PCMBS>PCMB. These results suggest that sulfhydryl groups are essential for dicarboxylate transport and that they may be located at or near substrate binding sites of the transporters in renal brush border and basolateral membranes.

• The Korean Journal of Physiology
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• v.30 no.1
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• pp.53-62
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• 1996

Transepithelial transport of tetraethylammonium (TEA) was studied in monolayers of opossum kidney cells cultured on permeable membrane filters. $[^{14}C]-TEA$ was transported across the OK cell monolayer from basolateral to apical side by a saturable process which can be stimulated by acidification of the apical medium. The apparent Michaelis-Menten constant $(K_{m})$ and the maximum velocity$(V_{max})$ for the transport were $41\;{\mu}M$ and 147 pmole/ mg protein/ min, respectively. The transport was significantly inhibited by unlabelled TEA, amiloride, cimetidine, choline, and mepiperphenidol added to the basolateral side at 1 mM and was slightly inhibited by 5 mM $N_{1}-methylnicotinamide\;(NMN).$ Unlabelled TEA added to the apical side stimulated the $basolateral-to-apical\;{^{14}C}-TEA$ transport, suggesting that the TEA self-exchange mechanism was involved at the apical membrane. Sulfhydryl reagents such as ${\rho}-chloromercuribenzoic\;acid\;(PCMB)\;and \;{\rho}-chloro-mercuribenzene\;sulfonate \;(PCMBS)$ and carboxyl reagents such as N,N'-dicyclohexylcarbodiimidem (DCCD) and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydro-quinoline(EEDQ) inhibited the TEA transport at both the basolateral and apical membranes of the OK cell monolayer. These results suggest that OK cell monolayers possess a vectorial transport system for organic cations which is similar to that for organic cation secretion in the renal proximal tubule.

• Korean Journal of Microbiology
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• v.19 no.1
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• pp.8-13
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• 1981

The content of sulfhydryl compounds in Chlorella cells during the life cycle in the synchronous culture is determined spectrophotomatically at 250nm(pH7.0) using p-CMB as SH-reagent. The changes in the content of-SHl compounds and protein in Chlorella cells is measured during the life cycle in connection with cell division and analyzed. 1) The amounts of total ninhydrin reactive substance increased with growth of cells but increased the more at the $L_4$ stage(cytokinesis stage) than at the $L_2$ stage (nuclear division stage). 2) The sulfhydryl content of Chlorella cells increased strikingly at the $L_2$ stage and decreased markedly at the $L_4$ stage. 3) The amounts of values -SH/protein showed a peak at the $L_2$ stage. The increase of the amount of total-SH compounds of cells during the nuclear division period was considered to be caused by the weighty roles of protein-SH groups for the formation of nuclear division apparatus and for the enzyme activity.

• BMB Reports
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• v.33 no.1
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• pp.43-48
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• 2000

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It converts the neurotransmitter 4-aminobutyric acid to succinic semialdehyde. In order to study the structural and functional aspects of catalytically active Cys residues of pig brain 4-aminobutyrate aminotransferase, we purified the active form in E. coli by coproduction of thioredoxin. The structural arrangement for functional requirements of a dimeric protein using a bifunctional sultbydryl reagent was then characterized, and the spatial proximity between the essential SH groups and a cofactor (pyridoxal-5'-phosphate) binding site was determined. The bifunctional sultbydryl reagent DMDS reacted with the enzyme at the ratio of one molecule per enzyme dimer. This resulted in an approximately 50% loss of enzymatic activity. The spatial proximity of the distance between the essential SH groups and the cofactor-binding site was determined by the energy transfer measurement technique. The result (approximate 20 ${\AA}$) suggested that cross-linking of two sulfhydryl groups with DMDS is not near a PLP binding site.

• The Journal of Korean Medicine
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• v.16 no.1 s.29
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• pp.281-294
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• 1995

The effect of Sam Hwa San on the Na-K-ATPase activity was evaluated in microsomal fraction prepared from rabbit cerebral cortex to determine whether Sam Hwa San affects Na-K-ATPase activity of nervous system. Sam Hwa San markedly inhibited the Na-K-ATPase activity in a dose-dependent manner with an estimated $I_{50}$ of 0.12%. Optimal pH for the Na-K-ATPase activity was at 7.5 in the presence or absence of Sam Hwa San. The degree of inhibition by the drug more increased at acidic and alkalic pHs than neutral pH. Kinetic studies of substrate and cationic activation of the enzyme indicate classic noncompetitive inhibition fashion for ATP, Na and K, showing significant reduction in Vmax without a change in Km. Dithiothreitol, a sulfhydryl reducing reagent, partially protects the inhibition of Na-K-ATPase activity by Sam Hwa San. Combination of Sam Hwa San and ouabain showed higher inhibition than cumulative inhibition. These results suggest that Sam Hwa San inhibits Na-K-ATPase activity in central nervous system by reacting with, at least a part, sulfhydryl group and ouabain binding site of the enzyme protein, but with different binding site from those of ATP, Na and K.

• Korean Journal of Microbiology
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• v.32 no.3
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• pp.222-231
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• 1994

Kinetic analysis was done to elucidate the reaction mechanism of purine nucleoside phosphorylase (PNP) in Saccharomyces cerevisiae. The binary complexes of PNP${\cdot}$phosphate and PNP${\cdot}$ribose 1-phosphate were involved in the reaction mechanism. The initial velocity and product inhibition studies demonstrated were consistent with the predominant mechanism of the reaction being an ordered bi, bi reaction. The phosphate bound to the enzyme first, followed by nucleoside and base were the first product to leave, followed by ribose 1-phosphate. The kinetically suggested mechanism of PNP in S. cerevisiae was in agreement with the results of protection studies against the inactivation of the enzyme by sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and 5,5'-dithiobisnitrobenzoate (DTNB). PNP was protected by ribose 1-phosphate and phosphate, but not by nucleoside or base, supporting the reaction order of ordered bi, bi mechanism. PCMB or DTNB-inactivated PNP was totally reactivated by dithiothreitol (DTT) and the activity was returned to the level of 77% by 2-mercaptoethanol, indicating that inactivation was reversible. The kinetic behavior of the PCMB-inactivated enzyme had been changed with higher $K_m$ value of inosine and lower $V_m$, and was restored by DTT. Inactivation of enzyme by DTNB showed similar pattern of K sub(m) value with that by PCMB, but had not changed the $V_m$ value, significantly. Negative cooperativity was not found with PCMB or DTNB treated PNP at high concentration of phosphate.

• Journal of the Korean Society of Food Science and Nutrition
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• v.19 no.4
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• pp.301-304
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• 1990

Factors affecting thermal inactivation and reactivation of korean radish peroxidase were inves-tigated,. The enzyme was stable below pH4.0 and above pH 8.0 The thermostablity of the enzyme was increased by addition of glucose sodium chloride and albuminl The inactivated enzyme by heat treatment was reactivated at room temperaturem The optimal pH for reactivation of the enzyme was pH of 9.0 The reactivation rate of the enyme was not afected by addition of glucose sodium chloride and albumin, The reactivation was completely inhibited by addition of sulfhydryl reagent such as dithiothreitol.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.1
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• pp.81-86
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• 1999

The factors affecting thermostability and reactivation of peroxidase from soybean sprouts(Glycine max L.) were investigated. The enzyme was the most stable at pH 7.0 and below 60oC. Thermostability of the enzyme was reduced by addition of sodium chloride and saccharides. The partially inactivated enzyme by heat treatment at 75oC for 10 min was reactivated up to 211.5% at the optimal reactivation condition. The optimal pH and temperature for reactivation of the enzyme were pH 9.0 and 40oC, respectively. The reactivation was completely inhibited by addition of sulfhydryl reagent such as L cysteine.

• BMB Reports
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• v.30 no.6
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• pp.403-409
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• 1997

Studies were undertaken to develop a competitive radioimmunoassay (RIA) and indirect antigen capture enzyme-linked immunosorbent assay (ELISA) for the determination of 5'-deoxy-5'-methylthioadenosine (MTA), which is formed from decarboxylated S-adenosylmethionine by spermidine and spermine synthase. Specific antiserum against MTA was raised in rabbits by immunization with MTA-BSA which was prepared by coupling BSA to oxidized MTA with periodate. Since MTA is oxidized easily to the sulfoxide, the sulfhydryl reagent, DTT. was added to the immunogen. For RIA, immunocomplexes were separated from free MTA by using ammonium sulfate precipitation. The antiserum showed almost no cross-reactivity with a variety of other nucleotides and riboses. But, the level of cross-reactivity of 5'-isobutylthioadenosine (SIBA) was high. These results showed the importance of hydrophobicity adjacent to the 5'-OH for determining antigenicity. The lower limit of detection by this assay was 100 fmol of MTA per tube. Using this assay. MTA levels were more easily and precisely determined in biological samples when compared with HPLC analysis. The RIA procedure is less time consuming. More than 24 analyses can be carried out in 2 h and required only a very small amount of sample ($20{\mu}l$ serum). In ELISA, biotin conjugated MTA-BSA was used as the labelled MTA. The sensitivity limit of this assay was lower than 100 pmol.