Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7.
Plant Physiol. 1987 May;84(1):19-24. doi: 10.1104/pp.84.1.19.
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 MgCl(2) 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 (LHCII(1):LHCII(3)) 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 LHCII(1):LHCII(3) 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 LCHII observed in situ and in vitro.
从经过冷驯化（RH）和未经冷驯化（RNH）的冬黑麦（Secale cereale L. cv Puma）中采用 JJ Burke、CL Ditto 和 CJ Arntzen（Arch Biochem Biophys 187: 252-263）的改良方法纯化光捕获复合物 II（LHCII）。通过用 Triton X-100 溶解类囊体膜，然后用 100 mM KCl 和 10 mM MgCl(2) 进行三次连续沉淀，基于叶绿素（Chl）基础上的产量最高可达 25%，基于多肽分析的纯度为 90%至 95%，在 4 小时内完成。多肽和色素分析、77 K 荧光发射和室温吸收光谱表明，通过这种改良方法获得的 LHCII 与通过其他已发表方法获得的 LHCII 相当。比较纯化的 RH 和 RNH LHCII 表明，在多肽、氨基酸、Chl 和类胡萝卜素组成以及脂质含量方面没有显著差异。然而，RH LHCII 与 RNH LHCII 仅在磷脂酰二酰甘油的脂肪酸组成上存在差异。RH LHCII 表现出与 PG 相关的 trans-Delta(3)-十六烯酸水平降低 54%，与 RNH LHCII 相比，寡聚 LHCII：单体 LHCII（LHCII(1):LHCII(3))降低 60%。RH 和 RNH LHCII 均表现出 PG 特异性的 5 倍富集。通过酶促水解完全去除 PG 导致 RH 和 RNH LHCII 的寡聚含量显著降低，使得 RH 和 RNH LHCII 制剂的 LHCII(1):LHCII(3)相同。这证实了这种特定的组成变化解释了在原位和体外观察到的 RH 和 RNH LCHII 之间的结构差异。
