TMAOase, trimethylamine-N-oxide demethylase, isa thermostable and active enzyme at 80°C

ABSTRACT:   Purified trimethylamine- N -oxide demethylase (TMAOase)from walleye pollack muscle is a thermostable protein that was notinactivated after heating at 80°C for 30 min.The heated enzyme was electrophoresed in the same manner as fornative enzyme. Circular dichroism (CD) spectra for purified enzymechanged reversibly in the temperature range of 10–80°C.As the enzyme was still active at 80°C, the CD spectralchange did not directly relate to enzyme activity. TMAOase activity inthe myofibrillar fraction decreased sharply above 30°C,but was extracted and recovered from the heated myofibrillar fraction,suggesting that the activity seemed to be interrupted and apparently inactivateddue to the thermal alteration of myofibrillar proteins or some unknownfactors. The complicated profile found in dimethylamine (DMA) formationfrom trimethylamine- N -oxide (TMAO) in walleye pollack muscleduring heating consisted of both enzymic and non-enzymic processes.Most DMA was produced enzymatically below 40°C and interruptedabove 40°C. Therefore, DMA and trimethylamine was formednon-enzymatically at high temperatures regardless of the presenceof native enzyme. A new, simple and easy purification method wasproposed based on the thermostable nature of the enzyme.     

敌草隆原药中二甲胺分析方法研究

本文建立了敌草隆原药中游离二甲胺的分析方法,先萃取后蒸馏的方法将二甲胺从敌草隆原药中分离出来,再采用酸碱返滴定的方法进行测定,该方法线性相关系数为0.999 1,标准偏差为0.007 5,变异系数为4.75%。回收率为97.86%~102.5%。该方法精密度高,准确度好,可以满足分析要求。     

Changes in metabolic profiles of urine from rats following chronic exposure to anticholinesterase pesticides

Previous studies have demonstrated that the anticholinesterase pesticides chlorpyrifos and carbaryl are neurotoxic to mammals. However, the toxicity of these pesticides to other organs and their potential interactive effects remain unclear. Our goal in this study was to assess the toxicities of ingestion of chlorpyrifos and carbaryl both separately, and in combination to non-nervous systems, especially the effect on urinary metabolic profiles, in rats. Chlorpyrifos, carbaryl and a mixture of these pesticides, were administered orally to Wistar rats for 90 consecutive days. Histopathological examination of liver and kidney and metabonomic analysis based on the urinary ¹H nuclear magnetic resonance spectra were used to investigate the toxic effects. The results showed that no histopathological changes were observed in the liver or kidney tissues, but metabonomic analysis revealed alternations in a number of urinary metabolites involving in the energy metabolism in liver mitochondria. Treatment of rats with chlorpyrifos alone led to an increase in creatine, glycine, dimethylglycine, dimethylamine, glutamine, succinate, alanine, lactate, and glucose. The categories of main differential urinary metabolites in carbaryl-treated rats were similar to those in chlorpyrifos-treated rats, whereas the changes were of varying degree. A combination of a low dose of chlorpyrifos and carbaryl resulted in an increase in the levels of main urinary metabolites compared to the controls, and the increase in signal intensity of the main metabolites was lower than that in the rats exposed to chlorpyrifos or carbaryl alone. All above results suggest that chronic exposure to chlorpyrifos and carbaryl alone, or in combination could cause disturbance of metabolic function in liver mitochondria and renal failure. Overall, we have shown that urine metabonomic analysis is non-invasive, sensitive, and relatively fast for assessing the individual or mutual effects following exposure to pesticides.