影响豌豆Rubisco组装的因子及其作用机制

The Factors Affecting Rubisco Assembly and the Reaction Mechanism in Pea

将新鲜制备的不含Rubisco的豌豆叶片低分子量 (LMW )蛋白组分在离体条件下于室温保温 4 8h后 ,经ND PAGE分析发现 ,ATP和Mg2 + 是组装的必需条件。随着Mg2 + 浓度的增加 ,组装成的Rubisco量逐渐增加 ,直至其浓度到 2 .5mmol/L后达到平衡。K+ 对组装有抑制作用 ,抑制程度随着K+ 浓度增加逐渐增强 ,当K+ 浓度为 5mmol/L时组装被抑制 5 6 % ,当K+ 浓度达到 4 0mmol/L时 ,组装被抑制高达 90 %以上 ;但同样浓度的Na+ 则没有抑制作用。另外 ,以羟自由基对豌豆LMW蛋白组分中的大亚基进行降解处理 ,对大亚基专一性降解肽谱与对全酶中的相同 ,也能产生 37kD的降解肽段。而且 ,预先经ATP和Mg2 + 处理的样品 ,LMW组分中 37kD的大亚基降解条带会消失 ,而在 2 0～ 30kD位置附近产生新的降解条带 ;而K+ 预处理同样能够产生 2 0～ 30kD大亚基的降解条带 ,但其 37kD的大亚基降解条带没有消失。这说明K+

The Rubisco deleted low molecular weight (LMW) protein components were obtained from pea leaves on Superose 6 column with FPLC, then incubated with ATP, K + or Mg 2+ at room temperature for 48 h. The results of ND PAGE analysis showed that ATP and Mg 2+ were necessary for Rubisco assembly (Fig.1), and the amount of Rubisco assembled increased with an increase in concentration of Mg 2+ in the assay medium up to 2.5 mmol/L (Fig.2). The process of assembly was inhibited apparently by the presence of K + (5 mmol/L) in the assay medium, and was inhibited almost completely(～90%) when the concentration of K + reached 40 mmol/L (Fig.3A). Assembly was not inhibited when K + was replaced by Na + at the same concentration(Fig.3B). On the other hand, when the LMW components incubated with ATP, Mg 2+ or K + respectively was treated with hydroxyl radical, the results of Western blot analysis after SDS PAGE indicated that the large subunit (LS) of Rubisco assembly intermediates could also be degraded into peptide of 37 kD (Fig.5 2)by hydroxyl radical just as the LS from holo enzyme (Fig.5 1). Furthermore, the fragmentation map of LS changed when LMW components were incubated with ATP and Mg 2+ before the hydoxyl radiacal treatment, and the molecular weight of degradation protein band became 20 30 kD instead of 37 kD(Fig.5 3,4). Similar result was obtained when LMW components were incubated with K + , but the degradation band of 37 kD was still there(Fig.5 5). It shows that K + is able to trigger the changes in LS conformation which hinders the assembly process.