| Part 1Preliminary identification of 3-DG ReductaseObjective To identify the 3-DG reductase extracted from chicken liver.Methods Enzyme activity was assayed by measuring the decrease in the absorbance at 340nm using 3-DG as substrate.Detection of the compound from 3-DG by treatment with the enzyme,mesurement of molecular weight of 3-DG reductase by SDS-Polyacrylamide Gel Electrophoresis, identification of 3-DG reducatse by Thin-Layer Chromatogram.Results The absorbance in reaction groups was significantly(p＜0.05) lower than that in control groups(0.649±0.107 vs 0.795±0.002,n=3),the enzyme showed two bands on SDS-Polyacrylamide Gel Electrophoresis, the band(49kDa) was estimated to be 3-DG reductase.The Rf value of the enzymatic reaction product and 3-DG on TLC were 0.52 and 0.6 respectively.Conclusion The molecular weight of the enzyme was estimated to be 49KDa,and the enzyme may convert 3-DG to 3-Deoxyfructose (3-DF).The chicken liver could be selected as an approriate source of stable access to 3-DG reductase.Part 2Determination of 3-DG in 3-DG Reductase enzymatic system in vitroObjective Two methods for the determination of 3-DG in 3-DG reductase enzymati -c system were developed for enzyme activity assessment.Methods With the 2,3-diaminon-aphthalene pre-column derivatization technique,3-DG was separated on a C18(0.46*25cm,5μm), the mobile phase was PBS-methol-methanol(70:5:25),the flow-rate was 1mL·min-1, fluorescence of each sample was determined at excitation/emission wavelengths of 271/503; enzymatic reaction sample was added to 2,4-dinitrophenylhydrazine-2mol·L-1 HCl,after incubation at 30℃for 30min the absorbance at 530 was measured.Results RP-HPLC:A good linearity was obtained in the range of 1～5μg·mL-1for 3-DG,the equation was Y=332.40x+34.50, r=0.9992,the average recoveries of 3-DG were 77.17%and the relative standard deviations were 8.98%(n=5);UV:A good linearity was obtained in the range of 1.28～17.91μg·mL-1 for 3-DG, the equation was Y=0.0382x-0.0002,r=0.9988,where Y is the concentration(μg·mL-1) and X is the absorbance at 530nm,the average recoveries of 3-DG were 109.93%and the relative standard deviations were 16.73%(n=6);significant correlation was observed between RP-HPLC and UV,r=0.86887(p＜0.001).Conclusion The two methods can be used to determine of 3-DG in 3-DG reductase enzymatic system.The two methods may serve as the basis of 3-DG reductase assessment. Part 3Research on the optimum condition,factors and evaluation extent of 3-DG reductase enzymatic reactionObjective Based on the established 3-DG reductase activity in enzymatic reaction in vitro described in part 2,optimization of the reaction conditions.Methods To identify the optimum enzymatic reaction condition,the enzyme was assayed with various concentration of NADPH,3-DG reductase,3-DG,time as well as the different temperature,pH,preserved condition and the co-enzyme(use NAD instead of NADPH),tested the effects of the influencing factors above on 3-DG reductase enzymatic reaction.Results The consumption of 3-DG was stable at NADPH concentration 0.15-0.20mg·mL-1 and 0.25-0.30mg·mL-1.Different 3-DG reductase volumes was added to the enzymatic system,the consumption of 3-DG showed a signi -ficant positive relationship with 3-DG concentration.Add the 3-DG reductase volumes more than 200μL,the consumption of 3-DG tend to be stable.The consumption of 3-DG showed an inverse correlation with 3-DG concentration.The consumption of 3-DG was stable between 60-120min.The consumption of 3-DG showed a positive correlation with temperature.The consumption of 3-DG was stable and high at pH7.4-7.9.We also tested the dehydrogenase activities of the enzyme purified by ammonium sulfate against 3-DG in the presence of NAD. The enzyme also showed dehydrogenase activity.The consumption of 3-DG was decreased rapidly after 30 day's preservation.The enzyme obtained by freezer drying was more stable than the other forms between the 30th and 180th day.Both the inhibitory effects of epalrestat and quercetin and the inducing effects of JZ01 on 3-DG reductase were observed.7002 had no effect on 3-DG reductase.Conclusion 3-DG reductase enzymatic activity could be assessed by the consumption of 3-DG.The optimum condition of 3-DG reductase enzymatic system was:the assay mixture contained in a total volume of 1mL,50 mmol·L-1PBS(pH7.4),100μL 2.5mg·mL-1 NADPH,100μL 1mg·mL-1 3-DG,100μL 3-DG reductase.The 3-DG reductase was easily affected by temperature,pH.It showed the dehydrogenase activities of the enzyme purified by ammonium sulfate against 3-DG in the presence of NAD but had no effect on 3-DG reductase. The enzyme activity was decreased after 30 day's preservation.Optimized 3-DG reductase enzymatic reaction system showed sensitive,accurate and stable and it may be a novel method in screening medicines. Part 4Preliminary application of 3-DG reductase system(treat and or prevent diabetic angiopathy)in vitroObjective To assess the 3-DG reductase activity in vitro for screening of medicine and Traditional Chinese medicine by using the optimized of 3-DG reductase enzymatic reaction system.Methods The enzyme was assayed with various concentration of glucose(5.5,12.5,25,50,100 mmol·L-1),epalrestat,aminoguanidine and quercetin were added to enzymatic reaction system respectively,after incubation at 30℃for 30min.Determination of the 3-DG concentration by RP-HPLC or UV in 3-DG reductase enzymatic reaction system.The 3-DG reductase activity was assessed by the consumption rate of 3-DG.Results The 3-DG reductase activity in 50,100 mmol·L-1 glucose group was significantly(p＜0.05) lower than that in reaction group,the enzymatic reaction was almost completely inhibited by treatment with epalrestat compared with reaction group,the 3-DG levels was significantly(p＜0.001) decreased in aminoguanidine group compared with reaction group,the enzymatic reaction was partial (p＜0.001) inhibited by treatment with quercetin compared with reaction group.Conclusion High concentration of glucose showed no inducing effect on 3-DG reductase,and the 3-DG reductase activity could be inhibited by 50 and 100 mmol·L-1 glucose concentration.Epalrestat which showed inhibitory effect on 3-DG reductase could inhibit the catabolism of 3-DG and lead to high level of 3-DG,and its inhibitory effect on 3-DG reductase may make a negative effect on therapeutic effect.Aminoguanidine react with 3-DG and had no effect on 3-DG reductase. Quercetin showed cetain inhibition on the 3-DG reductase.The effect of medicine on 3-DG reductase may be a new theraphy target. |
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