类囊体膜的提取

1、精选优质文档-倾情为你奉上浓度名称分子量捣碎液 : PH 7.8 2500ml 5000ml 1000ml20 mMTris-HCl121.14 (6.057g) （12.114） 2.42280.4 MSucrose342.3(342.3g) (684.6) 136.9215 mMNaCl58.44 (2.192g) (4.383) 0.87662 mMEDTA二钠 372.2 (1.861g) (3.722) 0.7444胀破液：PH 7.8 1000ml 2000ml20 mMTris-HCl121.14 (2.423g) (4.846)5 mMMgCl2203.3 (1.017g) (

2、2.033)15 mMNaCl58.44 (0.8766g) (1.753)保存液：PH 7.0 1000ml 2000ml20 mMTris-HCl121.14(2.423g) (4.846)35 mMNaCl58.44 (2.045) (4.09)0.4 MSucrose342.3 (136.92g) (273.84)Activities of electron transport: Thylakoid membranes were isolated from the leaves as described by Berthold et al. (1981). Whole chain el

3、ectron transport (H2O methyl viologen, MV) and partial reactions of photosynthetic electron transport mediated by PS2 (H2O2,6- dichloro-p-benzoquinone, DCBQ; H2Osilicomolybdate, SiMo) and PS1 (DCPIPH2MV) were measured as described by Nedunchezhian et al. (1997). Thylakoids were suspended at 10 mg(Ch

4、l) m-3 in the assay medium containing 20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 5 mM NH4Cl, and 100 mM sucrose supplemented with 500 M DCBQ and 200 M SiMo.类囊体的提取步骤：Highly active intact thylakoid membranes were prepared as in 6. Subchloroplast membranes capable of oxygen evolution were prepared

5、 by suspension of the intact thylakoids (2 mg chl/ml) in MgC12 (5 mM), NaCl(15 mM) and Hepes buffer (20 mM, pH 7.5) and incubation with Triton X-100 (25 mg/mg chl) at 4C for 30 min. The fraction of chl-containing material sedimenting at 40 000 X g (30 min) was resuspended in incubating buffer (2 mg

6、chl/ml) with Triton X-100 (5 mg/mg chl), recentrifuged immediately (40 000 X g, 30 min) and stored in sucrose (0.4 M), MgClz (5 mM), NaCl(15 mM) and Hepes (20 mM, pH 7.5) at -35C for subsequent analyses. In this procedure, which is superficially similar to that in 3, we have produced a set of condit

7、ions with regard to salt concentrations whereby O2 evolution activity is resistant to denaturation by exposure to Triton X-100. Procedures for assay of oxygen evolution, for Tris-inhibition, and EPR detection of signals II, and IIf have been reported in 7,8. Cytochrome content was assayed optically

8、by using an Aminco DW-2 spectrophotometer.Assay of pigments contentLeaf tissues were homogenized in chilled N, N-dimethylformamide using a mortar and pestle in dark at 4 C, and the homogenates were centrifuged at 8,800 g for 10 minutes. The supernatants were collected and the absorption spectra at 6

9、63 and 646 nm were recorded for the estimation of chlorophyll a (Chl a) and chlorophyll b (Chl b) following the procedure described by Inskeep and Bloom (1985). For the estimation of total carotenoids (Car), leaf tissues (300 mg) were homogenized in chilled 80% (v/v) acetone, and then centrifuged at 8,800 g for 10 minutes in dark at 4 C. The absorbanc