AN IN-VITRO NITRATE REDUCTASE ASSAY FOR MARINE MACROALGAE - OPTIMIZATION AND CHARACTERIZATION OF THE ENZYME FOR FUCUS-GARDNERI (PHAEOPHYTA)
AN IN-VITRO NITRATE REDUCTASE ASSAY FOR MARINE MACROALGAE - OPTIMIZATION AND CHARACTERIZATION OF THE ENZYME FOR FUCUS-GARDNERI (PHAEOPHYTA)
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DOI：
10.1111/j.0022-3646.1995.00835.x
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发表时间：
1995-10-01
影响因子：
2.9
通讯作者：
HARRISON, PJ
中科院分区：
文献类型：
--
作者：
HURD, CL;BERGES, JA;HARRISON, PJ
硝酸还原酶（NR）活性的测定可作为大型海藻氮代谢的一个有用指标。Silva，体外试验以前未能检测NR活性，需要使用原位（或所谓的“体内”）试验，其控制更松散，并且难以评估酶特性，如半饱和常数（K-m）。在本文中，我们描述了一种体外NR检测法，开发的F。gardneri，其中在测定之前使用液氮将组织匀浆。与以往的研究不同，在F. gardneri直接从田地中收集的硝酸盐转化为亚硝酸盐的水平高达30 nmol。min（-1）。g（-1）湿重。测试了通常添加到NR提取缓冲液中的各种化合物的效果。添加蛋白酶抑制剂，牛血清白蛋白，乙二胺四乙酸对NR活性没有一致的影响，而聚乙烯吡咯烷酮，铁氰化钾，黄素腺嘌呤二核苷酸显着降低活性。半饱和常数（Km）为NADH为0.18（+/-0.05）mM和硝酸盐，Km = 0.99（+/-0.41）mM。显着的NR活性检测没有添加硝酸盐，表明内部池的硝酸盐平均约20 μ mol NO3-。g（-1）湿重。2月的gardneri。NR活性在植株内的分布在个体之间差异很大，但菌柄中的活性比中部低约5倍。在从田间新鲜取样的植物中，NR活性从2月到3月增加了7倍，然后在4月下降到接近2月的水平。这些活动的变化可能与生长率的季节性变化相对应。该方法针对F. gardneri，用于来自不同分类群的几种不同的大型藻类物种：紫菜属，Coralina vagulveriensis Yendo，石莼属，Enteromorphaintegrifolia（Linnaeus）Nees、Macrocystisintegrifolia Bory和Costaria costatum（C.桑德斯。对于所有测试的物种，NR活性是可检测的，除了一个物种（紫菜属）。等于或大于其他工人在类似条件下对植物进行体内或体外测定所测得的活性。
Measurement of the activity of the enzyme nitrate reductase (NR) may provide a useful index of nitrogen metabolism in marine macroalgae, In several species, including Fucus gardneri P. C. Silva, in vitro assays previously failed to detect NR activity, necessitating the use of in situ (or so-called ''in vivo'') assays, which are more loosely controlled and bead to difficulties in assessing enzyme characteristics such as the half-saturation constant (K-m). In this paper, we describe an in vitro NR assay developed for F. gardneri, in which tissue was homogenized using liquid nitrogen prior to the assay. lit contrast to previous studies, enzyme activity was always detectable in F. gardneri collected directly from the field at levels up to 30 nmol nitrate converted to nitrite . min(-1). g(-1) wet weight. The effect of a variety of compounds, commonly added to NR extraction buffers, were tested. Additions of protease inhibitors, bovine serum albumin, and ethylenediamine tetraacetic acid had no consistent effects on NR activity, while polyvinyl pyrrolidone, potassium ferricyanide, and flavin adenine dinucleotide significantly decreased activity. The half-saturation constant (K-m) for NADH was 0.18 (+/-0.05) mM and for nitrate, K-m = 0.99 (+/-0.41) mM. Significant NR activity was detected without the addition of nitrate, suggesting that internal pools of nitrate averaging approximately 20 mu mol NO3-. g(-1) wet weight were present in F. gardneri in February. The distribution of NR activity within the plant was highly variable between individuals, but activities were approximately 5-fold lower in the stipe than in midregions. In plants freshly sampled from the field, NR activity increased 7-fold from February to March, then fell to near February levels by April. These changes in activity may correspond to seasonal changes in growth rate. The assay, optimized for F. gardneri, was used in several different macroalgal species from different taxa: Porphyra sp., Coralina vancouveriensis Yendo, Ulva sp., Enteromorpha intestinalis (Linnaeus) Nees, Macrocystis integrifolia Bory; and Costaria costatum (C. Agardh) Saunders. For all species tested, NR activity was detectable and, except for one species (Porphya sp.) was equal to or greater than activities measured by other workers using in vivo or in vitro assays for plants under similar conditions.