DEVELOPMENT AT COLD-HARDENING TEMPERATURES - THE STRUCTURE AND COMPOSITION OF PURIFIED RYE LIGHT HARVESTING COMPLEX-II
DEVELOPMENT AT COLD-HARDENING TEMPERATURES - THE STRUCTURE AND COMPOSITION OF PURIFIED RYE LIGHT HARVESTING COMPLEX-II
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DOI：
10.1104/pp.84.1.19
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发表时间：
1987-05-01
期刊：
影响因子：
7.4
通讯作者：
JAMES, DR
中科院分区：
文献类型：
--
作者：
KRUPA, Z;HUNER, NPA;JAMES, DR
采用JJ Burke， CL Ditto, CJ Arntzen （Arch Biochem biophyst187: 252-263）的改良工艺从冷硬化（RH）和未硬化的冬季黑麦（RNH） （Secale cereale L. cv Puma）中分离到光收获配合物II （LHCII）。Triton X-100溶解类囊体膜，然后用100 mM KCl和10 mM MgCl2连续沉淀三次，根据多肽分析，4小时内叶绿素（Chl）的产率高达25%，纯度为90 - 95%。多肽和色素分析、77 K荧光发射和室温吸收光谱表明，该方法得到的LHCII与其他已发表的方法得到的LHCII相当。纯化RH和RNH LHCII的比较表明，在多肽、氨基酸、Chl和类胡萝卜素成分方面没有显著差异，在脂质含量方面也没有差异。然而，RH LHCII与RNH LHCII仅在磷脂酰二酰基甘油的脂肪酸组成方面存在差异。RH LHCII的反式δ降低了54%。与PG相关的3-十六烯酸水平和低聚LHCII：单体LHCII （LHCII1:LHCII3）比RNH LHCII低60%。RH和RNH LHCII在PG中特异富集5倍。通过酶解完全去除PG导致RH和RNH LHCII的低聚物含量显著降低，使得RH和RNH LHCII制剂的LHCII1:LHCII3相同。这证实了这种特定的成分变化解释了原位和体外观察到的RH和RNH LHCII之间的结构差异。
Light harvesting complex II (LHCII) was purified from cold-hardened (RH) and nonhardened winter rye (RNH) (Secale cereale L. cv Puma) employing a modified procedure of JJ Burke, CL Ditto, CJ Arntzen (Arch Biochem Biophys 187: 252-263). Triton X-100 solubilization of thylakoid membranes followed by three successive precipitations with 100 mM KCl and 10 mM MgCl2 resulted in yields of up to 25% on a chlorophyll (Chl) basis and a purity of 90 to 95%, based on polypeptide analysis within 4 hours. Polypeptide and pigment analyses, 77 K fluorescence emission and room temperature absorption spectra indicate the LHCII obtained by this modified method is comparable to LHCII obtained by other published methods. Comparison of purified RH and RNH LHCII indicated no significant differences with respect to polypeptide, amino acid, Chl, and carotenoid compositions as well as no differences in lipid content. However, RH LHCII differed from RNH LHCII specifically with respect to the fatty acid composition of phosphatidyldiacylglycerol only. RH LHCII exhibited a 54% lower trans-.DELTA.3-hexadecenoic acid level associated with PG and a 60% lower oligomeric LHCII:monomeric LHCII (LHCII1:LHCII3) than RNH LHCII. Both RH and RNH LHCII exhibited a 5-fold enrichment in PG specifically. Complete removal of PG by enzymic hydrolysis resulted in a significant reduction in the oligomeric content of both RH and RNH LHCII such that LHCII1:LHCII3 of RH and RNH LHCII preparations were the same. This confirms that this specific compositional change accounts for the structural differences between RH and RNH LHCII observed in situ and in vitro.