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[交流] 求助酶活测定方法 PAL、CHS、F3H、DFR、LDOX

谁能提供以下植物中酶活性测定方法：

PAL、CHS、F3H、DFR、LDOX

谢谢了！

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1楼 2014-02-25 22:34:07

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2楼: Originally posted by sy9208 at 2014-09-15 14:32:02
LZ知道这些酶的测定方法了吗？我想知道CHS怎么测？

这个好像很难，我查到了2种方法
第一种：ASSAY OF CHALCONE SYNTHASE ACTIVITY BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY，PhytochemistVryo,l . 34,N o. 5, pp. 1225-1229，1993

第二种： CHS[12] chaleone synthase，查儿酮合成酶
Chemicals, [2-14C]Malonyl-CoA (1.71GBq•mmo1-1) was purchased from N.E.N. (Boston, Mass., USA) and [U-14C]phenyl-alanine (16.65GBq•mmol-1) from Amersham (Braunschweig, Germany). 4-Coumaroyl-CoA was synthesized by Dr. P.A. Bäumker (Institut für Botanik, Münster, Germany; Bäumker et al. 1988)[13].
Enzyme preparations. ö
Pbenylalanine ammonia-lyase: Leaves were cut into small pieces, frozen at -20°C and homogenized in a prechilled mortar in cold acetone (- 20°C The homogenate was filtered and the residue washed several times with cold acetone. The resulting dry powder was suspended in 0.1 M sodium borate buffer (pH 8.8) containing 5 mM dithioerythritol (DTE).
Chalcone synthase. All steps were carried out at 0-4°C Leaf material was homogenized in 0.1 M potassium phosphate buffer (pH 6.8) containing 40 mM ascorbate, 1 mM EDTA, 10 mM thiourea硫脲, 5 mM DTE and 0.25 g Polyclar AT.(g FW)-1 (Serva, Heidelberg, Germany). The homogenate was centrifuged for 10 min at 10 000 g and the supernatant concentrated by ultrafiltration (Diaflo PM 30; Amicon, Witten, Germany).
Enzyme assays, The activity of PAL was measured by the method of Amrhein and Zenk (1971). The activity of CHS was determined according to Beerhues and Wiermann (1988)[14], except that the pH of the incubation buffer was 7.8 and the final DTE concentration 0.5 mM. The analysis of the reaction products was performed as described by Lembach et al. (1989)[15].
(CHS activity was determined according to Beerhues and Wiermann (1985)[16]. The analysis of the reaction product was carried out using thin layer chromatography with silica gel plates and hexane: ethyl-acetate: methanol = 50:50:l (v/v/v) as a solvent system. For re-chromatography of the eluted product cellulose plates and 15% (v/v) ethanol were employed.)
Protein determination. Protein concentration was measured according to the method of Bradford (1976), as modified by Read and Northcote (1981), using ovalbumin as a standard.
Schöpker, H., M. Kneisel, L. Beerhues, et al. Phenylalanine ammonia-lyase and chalcone synthase in glands of Primula kewensis (W. Wats): immunofluorescence and immunogold localization. Planta, 1995. 196(4): 712-719.
13. PA, B., A. S and W. R. Metabolism of ferulic acid sucrose esters in anthers of Tulipa cv. Apeldoorn. II. Highly specific degradation of the esters by different esterase activities. Z. Naturforsch, 1988. 43c: 647-655.