Solubilization, partial purification, and characterization of a fatty aldehyde decarbonylase from a higher plant, Pisum sativum
Solubilization, partial purification, and characterization of a fatty aldehyde decarbonylase from a higher plant, Pisum sativum
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DOI：
10.1006/abbi.2000.1798
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发表时间：
2000-05-15
影响因子：
3.9
通讯作者：
Kolattukudy, P
中科院分区：
文献类型：
--
作者：
Schneider-Belhaddad, F;Kolattukudy, P
脂肪醛的酶促脱碳反应产生碳氢化合物。除绿藻外，任何生物体中都没有溶解和纯化出催化脱羰基的微粒酶。在这里，我们报道了从高等植物中提取的脱羧酶的增溶、纯化和部分性质。从豌豆叶片颗粒制剂中提取富含脱羧酶的脱羧酶，用P-辛基葡萄糖苷溶解，并进行部分纯化。SDS-PAGE显示在67 kDa处有一条主要蛋白条带。针对该蛋白的兔抗体与溶解的微粒体制剂中的67 kDa蛋白质发生交叉反应；抗核酮糖二磷酸羧基酶仅与该羧基酶的49 kDa大亚基发生交叉反应，而不与67 kDa附近的任何蛋白质发生交叉反应，表明绿藻酶制剂中没有发现任何来自交联大小亚基的羧基酶的污染。抗67-kDa蛋白抗体抑制了酶制剂催化的脱甲基化反应，表明该蛋白代表脱羧酶。纯化酶的脱羧酶活性需要磷脂才能发挥作用，磷脂酰胆碱是首选的脂类，磷脂酰丝氨酸和磷脂酰乙醇胺的替代效果较差。半峰活性在40MU十八烷基时达到最大。纯化的酶产生烷烃和CO，并被O-2、NADPH和DTE抑制。金属离子螯合剂对酶有严重的抑制作用，而Cu2+则完全恢复了酶的活性。纯化的酶制剂始终显示铜的存在，而铜原卟啉IX催化脱羧反应。这些结果表明，这种高等植物酶可能是一种铜酶，而不是被发现有Co.(C)2000学术出版社的绿藻酶。
Enzymatic decarbonylation of fatty aldehydes generates hydrocarbons. The particulate enzyme that catalyzes the decarbonylation has not been solubilized and purified from any organism but a green alga. Here we report the solubilization, purification, and partial characterization of the decarbonylase from a higher plant. Decarbonylase from a particulate preparation from pea (Pisum sativum) leaves, enriched in decarbonylase, was solubilized with P-octyl glucoside and partially purified. SDS-PACE showed a major protein band at 67 kDa. Rabbit antibodies raised against this protein specifically cross-reacted with the 67-kDa protein in solubilized microsomal preparations; anti-ribulose bisphosphate carboxylase cross-reacted only with the 49-kDa large subunit of the carboxylase, but not with any protein near 67 kDa, showing the absence of any contamination from cross-linked small-large subunit of the carboxylase found in the green algal enzyme preparation. Anti-67-kDa protein antibodies inhibited decarbonylation catalyzed by the enzyme preparations, showing that this protein represents the decarbonylase. Decarbonylase activity of the purified enzyme required phospholipids for activity; phosphatidylcholine was the preferred lipid although phosphatidylserine and phosphatidylethanolamine could substitute less effectively. Half-maximal activity was observed at 40 mu M octadecanal. The purified enzyme produced alkane and CO and was inhibited by O-2, NADPH, and DTE. Metal ion chelators severely inhibited the enzyme and Cu2+ fully restored the enzyme activity. Purified enzyme preparations consistently showed the presence of Cu, and copper protoporphyrin IX catalyzed decarbonylation. These results suggest that this higher plant enzyme probably is a Cu enzyme unlike the green algal enzyme that was found to have Co. (C) 2000 Academic Press.