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Preparation of extracts from mature spruce needles for enzymatic analyses
Author(s) -
Weimar Maria,
Rothe Gunter M.
Publication year - 1987
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1987.tb01986.x
Subject(s) - dehydrogenase , chemistry , chromatography , enzyme , glutamate dehydrogenase , malate dehydrogenase , biochemistry , sephadex , phosphogluconate dehydrogenase , substrate (aquarium) , glucose 6 phosphate dehydrogenase , biology , glutamate receptor , ecology , receptor
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 m M 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.