• Bulletin of the Korean Chemical Society
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• 제21권12호
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• pp.1217-1221
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• 2000

L-alanine dehydrogenase from Bacillus subtilis exhibits allosteric kinetic properties in the presence of $ZN^{2+}$. $ZN^{2+}$ induces the binding of substrate (L-alanine) to be cooperative at pH 8.0. The effect of pH variation between pH 7.0 and pH 10.0 on the inhibition by $ZN^{2+}$ correlates with the pH effect on the $K_m$ values for L-alanine within these pH range indicating that $ZN^{2+}$ and substrate compete for the same site. No such cooperativity is induced by $ZN^{2+}$ when the reaction is carried out at pH 10. At this higher pH, $ZN^{2+}$ binds with the enzyme with lower affinity and noncompetitive with respect to L-alanine. Inhibition of L-alanine dehydrogenase by $ZN^{2+}$ depends on the ionic strength. Increase in KCI concentration reduced the inhibition, but allosteric property in $ZN^{2+}$ binding is conserved. A model for the regulatory mechanism of L-alanine dehydrogenase as a noncooperative substrate-cooperative cofactor allosteric enzyme, which is compatible in both concerted and the sequential allosteric mechanism, is proposed.

• 분석과학
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• 제8권3호
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• pp.359-364
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• 1995

Urinary dipeptidase와 alanine dehydrogenase의 연쇄반응을 이용한 형광분석법을 개발하였다. 반응계는 기질로서 L-ala-ala, ${\beta}-NAD^+$, L-alanine dehydrogenase와 pH 9의 12.5mM sodium carbonate buffer를 포함하며 urinary dipeptidase를 가함으로써 반응을 시작했다. 생성된 NADH는 여기파장 340nm, 형광파장 460nm에서 측정했다. 기존의 glycyldehydrophenylalanine(Gdp)의 가수분해 방법과 형광분석법을 비교한 결과 0.996의 높은 상관계수를 나타냈으며 10배 이상의 감도 증가를 보였다.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.628-631
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• 2003

The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to $70^{\circ}C$. The activity of the enzyme was inhibited by 1 mM $Zn^{2+}$, 20% hexane, and 20% $CHCl_3$. However, 10 mM $Mg^{2+}$ and 40% propanol had no effect on the enzyme activity. The Michaelis constants ($K_m$) for the substrates were $50\;\mu\textrm{M}$ for NADH, 0.2 mM for pyruvate, 39.4 mM for $NH_4+$, 2.6 mM for L-alanine, and 1.8 mM for $NAD^+$.

• BMB Reports
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• 제31권1호
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• pp.25-30
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• 1998

Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of $Zn^{2+}$ were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when $Zn^{2+}$ is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the xinc ion concentration: the Hill coefficients ($n_H$) varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to $60\;{\mu}m$. The inhibition of AlaDH by $Zn^{2+}$ is reversible and noncompetitive with respect to $NAD^+$ ($K_i\;=\;5.28{\times}10^{-5}\;M$). $Zn^{2+}$ itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of $Zn^{2+}$ are not affected by the enzyme concentration indicating that $Zn^{2+}$ binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, $Zn^{2+}$ appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that $Zn^{2+}$ is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the $Zn^{2+}$ from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDh from Bacillus subtilis is consistent with both MWC and Koshland's allosteric model.

• 대한의생명과학회지
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• 제20권3호
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• pp.124-128
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• 2014

Alcoholism is a significant global health problem. Alcohol dehydrogenase and aldehyde dehydrogenase play important roles in the metabolism of alcohol and aldehyde. In this study, we aimed to investigate the eliminatory effects of a fruit extract drink on alcohol metabolism in drunken Sprague-Dawley (SD) rats. Male SD rats were given a fruit extract drink or a commercial product (10 mL/kg) 30 min prior to 40% (5 g/kg) ethanol ingestion. To assay the effect of the fruit extract drink on blood ethanol concentration, blood samples were taken from the saphenous vein at 3 and 5 h after ethanol ingestion. The blood concentrations of alcohol, alcohol dehydrogenase, and aldehyde dehydrogenase were significantly lower in the fruit extract drink group than in the control group, in a time-dependent manner. However, the alanine aminotransferase and aspartate aminotransferase activities of all experimental groups were unaltered compared to those of the control group. These results suggested that fruit extract drink intake can have a positive effect on the reduction of alcohol, alcohol dehydrogenase, and aldehyde dehydrogenase concentrations in the blood and may alleviate acute ethanol-induced hepatotoxicity by altering alcohol metabolic enzyme activities.

• 한국응용과학기술학회지
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• 제36권4호
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• pp.1420-1426
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• 2019

Dawley 계 숫컷 흰쥐(200~210g)를 정상군, CCl₄-대조군, CCl₄-실험군으로 나누어 실험군은 애엽 에탄올 추출물을 1,000mg/kg,b.w의 용량으로 1일1회7일간 경구 투여 후 사염화탄소를 0.6mg/kg,b.w의 용량으로 복강내 주사 후 다음날 개복하여 혈청내의 Alanine aminotransferase(ALT), Aspartate aminotransferase(AST),Alkaline phosphatase(ALP), Glutamyltranspeptidase(γ-GT), Lactate dehydrogenase(LDH)의 활성도와 중성지방, 콜레스테롤 함량을 측정한 결과 애엽 추출물 투여군에서 CCl₄-대조군과 비교하여 유의적인 감소를 나타내었으며, HDL-콜레스테롤은 유의적인 증가를 나타내었다. 간조직중의 지질과산화 함량은 감소를 glutathione함량은 유의적인 증가를 나타내었다 이 실험 결과 애엽 에탄올 추출물은 사염화탄소 투여에 의한 간 손상을 억제하는 보호물질을 함유하고 있음을 알 수 있었다.

• 한국축산식품학회지
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• 제39권1호
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• pp.13-22
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• 2019

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in Lactobacillus johnsonii PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the ${\beta}$-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into E. coli-Lactobacillus shuttle vector pULP2, which was generated by the fusion of pUC19 with the L. plantarum plasmid pLP27. Finally, the constructed vectors were introduced into L. plantarum for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in L. plantarum until 80 generations without selection pressure. A bile-responsive expression vector, $pULP3-P_{LDH}$, for Lactobacillus spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.

• Journal of Applied Biological Chemistry
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• 제56권2호
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• pp.89-93
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• 2013

Gamma-aminobutyric acid (GABA) aminotransferase (gabT) and succinic semialdehyde dehydrogenase (gabD) genes from Pseudomonas fluorescens KCCM 12537 were cloned into a single pETDuet-1 vector and co-expressed in Escherichia coli BL21(DE3) simultaneously. The mixture of both enzymes, called GABase, is the key enzyme for the enzymatic analysis of GABA. The molecular mass of the GABA aminotransferase and succinic semialdehyde dehydrogenase were determined to be 52.8 and 46.7 kDa following computations performed with the pI/Mw program, respectively. The GABase activity between pH 6.0 and 9.0 for 24 h at $4^{\circ}C$ remained over 75%, but under pH 6.0 decreased rapidly. The GABase activity between 25 and $35^{\circ}C$ by the treatment at pH 8.6 for 30 min remained over 80%, but over $35^{\circ}C$ decreased rapidly. When the activity against GABA was defined as 100%, the purified GABase activity against 5-aminovaleric acid having a similar structure to GABA showed 47.7% and GABase activity against ${\beta}$-alanine, ${\varepsilon}$-amino-n-caproic acid, $_L$-ornithine, $_L$-lysine, and $_L$-aspartic acid showed between 0.3 to 2.3%. The GABA content was analyzed with this co-expressed GABase, compared with the other GABase which was available commercially. As a result, the content of GABA extracted from brown rice, dark brown rice, and black rice were $26.4{\pm}3.5$, $40.5{\pm}4.7$ and $94.7{\pm}9.3{\mu}g/g$, which were similar data of other GABase in the error ranges.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권1호
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• pp.59-68
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• 2007

Changes in protein metabolism were studied in hemolymph and fat body on days 1, 3, 5 and 7 of the fifth-instar silkworm, Bombyx mori, exposed to lethal, sublethal doses and prevailing levels of fluoride in groundwater in Karnataka and Andhra Pradesh States of India. The total protein content indicated a depletion followed by a concomitant increase in accumulation of free amino acids. Concurrently, the activity of protease in both of the tissues was also increased. A steady enhancement in the activities of alanine aminotransferase and aspartate aminotransferase paralleled the elevation of glutamate dehydrogenase activity in the tissues studied. It is presumed, on the basis of these results, that the fluoride toxicity causes major changes in protein metabolism of the silkworms.

• Food Science and Biotechnology
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• 제18권6호
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• pp.1411-1416
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• 2009

The suppressive effects of glutathione-enriched Saccharomyces cerevisiae FF-8 strain (FF-8 GY) on alcoholinduced hepatotoxicity have been studied. FF-8 GY (256 mg/L) from the fermentation at a large scale bioreactor was used. Either of 5% FF-8 GY or 5% commercial glutathione-enriched yeast extract (GYE) with or without 30% alcohol was tested with rats for 4 weeks. FF-8 GY and GYE were found to reduce those alcohol-elevated serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) activities. Blood alcohol and acetaldehyde were also decreased by FF-8 GY and GYE. Interestingly, FF-8 GY drastically increased both hepatic alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities in comparison to GYE group, thus FF-8 GY would be more effective in blood alcohol and acetaldehyde reduction. Attenuated lipid droplet accumulation in hepatocytes was observed in both FF-8 GY and GYE when alcohol stimulated the accumulation. Therefore, FF-8 GY may be useful to protect liver from alcohol-induced hepatotoxicity.