Preparation of extracts from mature spruce needles for enzymatic analyses
Preparation of extracts from mature spruce needles for enzymatic analyses
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成熟云杉针叶提取物的制备用于酶分析
DOI：
10.1111/j.1399-3054.1987.tb01986.x
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发表时间：
1987
影响因子：
6.4
通讯作者：
G. Rothe
中科院分区：
文献类型：
--
作者：
M. Weimar;G. Rothe
从云杉针叶中同时提取参与碳水化合物或氨基酸代谢的几种活性酶是可能的。喀斯特。]当a)使用含有5%PVPP和0.5%Triton X-100的100 mM Na-PI缓冲液(含5%PVPP和0.5%Triton X-100)，以及当b)用FractoGel TSK HW-40分离介质进行凝胶层析时，所得粗提物从低分子化合物中去除。除Triton X-100外，还检测了Triton X-305、Myrij-52和Brij-35，但0.5%的Triton X-100提取的酶提取物活性最高。在初夏从云杉针叶制备的粗提物中，当加入共底物NADP+时，在334 nm处的吸光度显著增加，因此无法进行可靠的分光光度分析。用凝胶层析法可消除干扰小分子物质。以Bio-Gel P-6 DG、Sephadex G-25M、Trisacryl GF 05和FractoGel TSK HW-40(F)为分离介质，以FractoGel的活性最高。
测定了葡萄糖-6-磷酸脱氢酶(EC 1.1.1.49)、6-磷酸葡萄糖脱氢酶(EC 1.1.1.44)、葡萄糖-6-磷酸异构酶(EC 5.3.1.9)、莽草酸脱氢酶(EC 1.1.1.25)、NAD+-苹果酸脱氢酶(EC 1.1.1.37)、谷氨酸脱氢酶(EC 1.4.1.2)、天冬氨酸氨基转移酶(EC 2.6.1.1)和丙氨酸转氨酶(EC 2.6.1.2)的活性。NAD+-苹果酸脱氢酶和6-磷酸葡萄糖酸脱氢酶的活性估计分别与白云杉和樟子松针叶酶的活性范围相同。
It was possible to extract simultaneously several active enzymes involved in the carbohydrate or the amino acid metabolism from spruce needles [Picea abies (L.) Karst.] when a) a 100 mM Na-Pi buffer of pH 7.5 containing 5% PVPP and 0.5% Triton X-100 was used and when b) the resulting crude extracts were freed from lowmolecular-weight compounds by gel-chromatography using the separation medium Fractogel TSK HW-40. Besides Triton X-100, Triton X-305, Myrij-52 and Brij-35 were tested, but 0.5% Triton X-100 brought about the most active enzyme extracts. In crude extracts prepared from spruce needles during the early summer a high increase in absorbance at 334 nm was observed when the co-substrate NADP+ was added, thus making reliable spectrophotometric assays impossible. The interfering low-molecular-weight substances could be eliminated by gel chromatography. As separation media Bio-Gel P-6 DG, Sephadex G-25 m, Trisacryl GF 05 and Fractogel TSK HW-40 (F) were tested, with Fractogel yielding the highest activities.
With the methods described in this paper the activities of the following enzymes were determined: glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate isomerase (EC 5.3.1.9), shikimate dehydrogenase (EC 1.1.1.25), NAD+-malate dehydrogenase (EC 1.1.1.37), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2). The activities estimated for NAD+-malate dehydrogenase and 6-phosphogluconate dehydrogenase are in the range of those published for the needle enzymes of white spruce and Scots pine, respectively.