Determination of Fructokinase Activity from Zobellia galactanivorans
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Author:
Date:
2015-11-05
[Abstract] Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g.,, the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study ...
[摘要] 甘露醇是在多种活生物体中发生的多元醇,其中它实现不同的生理作用。已经描述了用于由细菌代谢甘露醇的几种途径,包括磷酸烯醇丙酮酸依赖性磷酸转移酶系统(PST)和基于M2DH的分解代谢途径。后者涉及两种酶,甘露醇-2-脱氢酶(EC 1.1.1.67)和果糖激酶(EC 2.7.1.4),并且已经在不同的细菌中鉴定,例如海洋拟杆菌Zobellia galactanivorans ( Zg ),其最近获得了研究大分子多糖降解的兴趣。该方案描述了Zobellia 半乳糖厌氧杆菌的重组果糖激酶(FK)的生物化学表征。 < strong> [原理] 果糖激酶(FK)活性通过以下方法确定:果糖激酶(FK)活性通过使用ATP作为辅因子来催化果糖转化成果糖-6-磷酸。酶偶联测定(图1)。通过FK反应(即果糖磷酸化成果糖-6-磷酸(F6P))形成的ADP由将磷酸烯醇丙酮酸(PEP)转化为丙酮酸的丙酮酸激酶(PK)使用。然后,乳酸脱氢酶(LDH)使用NADH作为辅因子将丙酮酸转化为乳酸。通过跟随在340nm处的吸光度的降低来测量FK活性,其对应于NADH转化为NAD + 。 ...
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Determination of Mannitol-2-dehydrogenase Activity
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Author:
Date:
2015-11-05
[Abstract] Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g., the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study ...
[摘要] 甘露醇是在多种活生物体中发生的多元醇,其中它实现不同的生理作用。已经描述了用于由细菌代谢甘露醇的几种途径,包括磷酸烯醇丙酮酸依赖性磷酸转移酶系统(PST)和基于M2DH的分解代谢途径。后者涉及两种酶,甘露醇-2-脱氢酶(EC 1.1.1.67)和果糖激酶(EC 2.7.1.4),并且已经在不同的细菌中鉴定,例如, 海洋拟杆菌(Zobellia galactanivorans)( Zg ),最近对研究大分子多糖降解有兴趣。该方案描述了半乳聚糖微生物的重组甘露醇-2-脱氢酶(M2DH)的生物化学表征。使用NAD +辅助作为辅因子,M2DH酶催化甘露醇向果糖的可逆转化。 在两个方向上测定M2DH活性,即果糖还原和甘露醇氧化。 可逆反应: D-甘露醇+ NAD + ↔D-果糖+ NADH + H +
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Optical Clearing Using SeeDB
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Author:
Date:
2014-02-05
[Abstract] We describe a water-based optical clearing agent, SeeDB (See Deep Brain), which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. SeeDB is a saturated solution of fructose (80.2% w/w) in water with 0.5% α-thioglycerol. In standard SeeDB optical clearing procedure, we treat paraformaldehyde-fixed embryo and brain samples with increasing concentrations of aqueous fructose solutions, and finally equilibrate them in SeeDB. The entire procedure takes approximately three days. Unlike previous ...
[摘要] 我们描述了一种水基光学清除剂,SeeDB(参见深脑),其在几天内清除固定的脑样品,而不淬灭许多类型的荧光染料,包括荧光蛋白和亲脂性神经元示踪剂。 SeeDB是果糖(80.2%w/w)在含有0.5%α-硫代甘油的水中的饱和溶液。在标准的SeeDB光学清除程序中,我们用增加浓度的果糖水溶液处理多聚甲醛固定的胚胎和脑样品,并且最后在SeeDB中平衡它们。整个过程大约需要三天。与以前的方法不同,该方法在清除过程中保持恒定的样品体积,这是保持细胞形态完整的重要因素。光学清除后,我们可以达到>在共聚焦显微镜下为1,000μm。当与双光子显微镜结合时,SeeDB允许我们以毫米级别水平成像固定的小鼠大脑。这种方法有助于全面和定量分析理解神经元电路,在成人和发展中国家的小鼠大脑。还提供了SeeDB变体(参见DB37)和优化的程序(见DBp和SeeDB37ht协议)以满足特定要求。
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