Hagopian Kevork
University of California, School of Veterinary Medicine, Department of Molecular Biosciences, Davis 95616, USA.
Ital J Biochem. 2007 Mar;56(1):6-17.
Xanthine oxidase (XO) and total oxidase plus dehydrogenase (XO+XDH) activities from rat liver were measured in the presence or absence of adenine in extracts prepared with or without DTT/PMSF in homogenization buffer. Presence of adenine in extracts, prepared with or without DTT/PMSF, caused a 45-60% decrease in XO and XO+XDH activities. Removal of adenine by dialysis from extracts prepared with or without DTT/PMSF resulted in the recovery of XO and XO+XDH activities to almost their pre-dialysis control levels. Enzyme activity after 24hr storage at -20 degrees C depended on the presence or absence of DTT/PMSF and adenine, with both XO and XO+XDH activities being lower in extracts with the combined presence of DTT/PMSF and adenine. Incubation of extracts at 37 degrees C for 30 minutes resulted in increased XO and XO+XDH activities, however, adenine-treated samples did not differ from their pre-incubation activities. The molecular mass of the enzyme from control and adenine-treated extracts was unchanged (300 kDa). Adenine-treated extracts prepared with or without DTT/PMSF showed higher D/O ratios in all post-dialysis samples when compared with their pre-dialysis ratios. The results suggest that adenine may play a role in preventing the dehydrogenase to oxidase conversion during extract preparation, storage, overnight dialysis and heat treatment.
在含有或不含二硫苏糖醇（DTT）/苯甲基磺酰氟（PMSF）的匀浆缓冲液中制备大鼠肝脏提取物，在有或无腺嘌呤存在的情况下，测定黄嘌呤氧化酶（XO）以及总氧化酶加脱氢酶（XO+XDH）的活性。无论提取物制备过程中是否含有DTT/PMSF，腺嘌呤的存在都会使XO和XO+XDH的活性降低45%-60%。通过透析从含有或不含DTT/PMSF的提取物中去除腺嘌呤后，XO和XO+XDH的活性恢复到几乎透析前的对照水平。在-20℃储存24小时后的酶活性取决于DTT/PMSF和腺嘌呤的存在与否，在同时存在DTT/PMSF和腺嘌呤的提取物中，XO和XO+XDH的活性均较低。将提取物在37℃孵育30分钟会使XO和XO+XDH的活性增加，然而，经腺嘌呤处理的样品与其孵育前的活性没有差异。对照提取物和经腺嘌呤处理的提取物中酶的分子量没有变化（300 kDa）。与透析前的比率相比，无论是否含有DTT/PMSF，经腺嘌呤处理的提取物在所有透析后的样品中均显示出更高的D/O比率。结果表明，腺嘌呤可能在提取物制备、储存、过夜透析和热处理过程中防止脱氢酶向氧化酶的转化中发挥作用。
