Regulation of xanthine oxidase in rat liver: modifications of the enzyme activity of rat liver supernatant on storage at 20 degrees.
Regulation of xanthine oxidase in rat liver: modifications of the enzyme activity of rat liver supernatant on storage at 20 degrees.
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大鼠肝脏黄嘌呤氧化酶的调节：20度保存的大鼠肝脏上清液酶活性的改变。
DOI：
10.1042/bj1080349
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发表时间：
1968
期刊：
影响因子：
--
通讯作者：
F. Stirpe
中科院分区：
文献类型：
--
作者：
E. Corte;F. Stirpe
图1.在-20℃保存的大鼠肝脏上清液的黄嘌呤氧化酶活性。为了避免重复解冻，分离的样品保持在-20℃，并在化验前立即解冻。试剂盒的最终体积为3-0ml.Olm-tris-HCI缓冲液，pH8*1，60，微黄嘌呤和0-2ml.大鼠肝脏上清液。黄嘌呤氧化酶活性以每分钟形成的尿酸分子数来表示。在6到20小时之间完成，不同的制剂有很大的差异，活性按照S型曲线增加(图1)。提取物的活性保持在-200，然后在几天内保持稳定。在“活化”之前，NAD在黄嘌呤的氧化过程中被降低，如反应混合物的E340增加所表明的那样。在NAD存在下形成的尿酸的量大约等于在02存在下形成的尿酸加上NAD减少的量的总和(三个实验的平均值，单位为m，umoles/min：由02形成的尿酸，2×5，其中NAD为8-7；形成NADH2，7.4)。这似乎表明NAD是在其存在时被氧化的额外数量的黄嘌呤的受体。在“活化”后，几乎没有观察到NADH2的形成(相同样品的值与上面相同，结果的顺序相同：9*0，8*2和0.5)。人们试图通过其他方式获得“激活”：快速冷冻和解冻提取物，重复多达四次，导致酶活性相当轻微的损失。如果将上清液在00℃保持5小时，或在370℃孵育1小时，或在500℃加热4分钟，则只有部分激活，仍未完成。在-200℃下获得的“活化”不会被逆转，如果“活化的”提取物在制备过程中被再次透析。被激活的和未被激活的样品的等量混合的酶活性始终是个体数值的平均值。用鸡肝上清液进行了类似的实验，但当这些制剂储存在-20℃时，没有观察到黄嘌呤脱氢酶活性的改变。Di8cu8ion.当大鼠肝脏上清液储存在-200℃时，黄嘌呤氧化酶与02受体的反应速度加快，由此可以推断，在新制成的制剂中，该酶对02的亲和力较低，或者只有一小部分酶可能与02反应。我们的实验还表明，大鼠肝脏黄嘌呤氧化酶可能与NAD发生反应。在这方面，该酶和鸡肝的黄嘌呤脱氢酶之间存在差异，因为后者与NAD的反应速度大致相同(Morell，1955)或更高(E.Della Corte和F.Stirpe，未发表的工作)，而在我们的大鼠肝脏非激活制剂中，与NAD的反应速度约为与亚甲基蓝的反应速度的一半。在NAD存在下反应的化学计量表明，黄嘌呤在“活化”之前，部分被02氧化，部分被NAD氧化。在“激活”后没有观察到这样的对应关系；这目前无法解释，但在这些条件下缺乏可检测到的NADH_2导致了以下假设：(I)与02的反应干扰了与NAD的反应，或(Ii)形成的NADH_2被黄嘌呤氧化酶氧化(NADH_2是这种酶的底物；Corran，Dewan，Gordon&Green，1939；Mackler，Mahler&Green，1954)，或(Iii)酶与NAD反应的能力丧失。已有不同温度下酶活性随时间增加的报道。从猪肝脏中提纯的黄嘌呤氧化酶在700℃短时加热后活性增加(Murray&Chaykin，1966)；…的活性
Fig. 1. Xanthine oxidase activity of rat liver supernatant stored at-20. To avoid repeated thawing, separate samples were kept at-20 and thawed immediately before the assay. The assay mixture contained, in a final volume of 3-0ml., OlM-tris-HCI buffer, pH8* 1, 60, uM-xanthine and 0-2ml. of rat liver supernatant. Xanthine oxidase activity is expressed as mjumoles of uric acid formed/min. completion between 6 and 20hr., with considerable variation from a preparation to another, the activity increasing according to a sigmoidal curve (Fig. 1). The activity of the extracts kept at-200 then remained stable for several days. Before'activation'NAD was reduced during the oxidation of xanthine, as shown by the increased E340 of the reaction mixtures. The amount of uric acid formed in the presence of NAD was approxi-mately the sum, in molar terms, of the uric acid formed in the presence of 02 plus the amount of NAD reduced (mean values of three experiments, in m, umoles/min.: uric acid formed with 02, 2* 5, with NAD, 8-7; NADH2 formed, 7.4). This seems to indicate that NAD is the acceptor for the extra amount of xanthine oxidized in its presence. After'activation'almost no formation of NADH2 was observed (values of the same samples as above, in the same order of results: 9* 0, 8* 2 and 0.5). Attempts were made to obtain the'activation'in other ways: rapid freezing and thawing of the extracts, repeated up to four times, caused rather a slight loss of enzyme activity. If the supernatants were kept at 00 for up to 5hr., or incubated at 370 for up to 1hr., or warmed at 500 for 4min., therewas only a partial'activation', which did not goto completion. The'activation'obtained at-200 was not reversed if the'activated'extracts were dialysed againas practised duringtheir preparation. The enzyme activities of mixtures of equal parts of'activated'and non-activated samples were consistently the average of individual values. Similar experiments were performed with chick liver supernatants, but no modifications of the xanthine dehydrogenase activity were observed when these preparations were stored at-20. Di8cus8ion. The rate of the xanthine oxidase reaction with 02 as acceptor increases when rat liver supernatants are stored at-200, and from this it may be inferred that in freshly made preparations either the enzyme has a lower affinity for 02, or only a minor part of it may react with 02. Our experiments indicate also that rat liver xanthine oxidase may react with NAD. A difference exists, in this respect, between this enzyme and the xanthine dehydrogenase of chick liver, since the latter reacts at approximately the same (Morell, 1955) or at a higher rate (E. Della Corte & F. Stirpe, unpublished work) with NAD than with methylene blue, whereas in our non-activated preparations from rat liver the reaction rate with NAD is about one-half of that with methylene blue. The stoicheio-metry of the reaction in the presence of NAD shows that before'activation'xanthine is oxidized in part by 02 and in part by NAD. Such a corre-spondence is not observed after'activation'; this cannot be explained at the moment, but the lack of detectableNADH2underthese conditions leadsusto suppose either (i) that the reaction with 02 interferes with that with NAD, or (ii) that the NADH2 formed is reoxidized by xanthine oxidase (NADH2 is a substrate for this enzyme; Corran, Dewan, Gordon & Green, 1939; Mackler, Mahler & Green, 1954), or (iii) that the capacity of the enzyme to react with NAD is lost.Increases of enzyme activities with time at various temperatures have been reported. The activity of xanthine oxidase purified from pig liver is increased after short heating at 700 (Murray & Chaykin, 1966); the activity of …