Purification and Characterization of a Low-Molecular-Weight Phospholipase A(2) from Developing Seeds of Elm

Phospholipase A(2) (PLA(2)) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca(2+) for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA(2), neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA(2)s and, to our knowledge, is the first plant enzyme of this type to be described.

磷脂酶A₂（Phospholipase A₂, PLA₂）的纯化倍数相较于提取自欧洲白榆（Ulmus glabra）发育中种子的初始可溶性蛋白提取物，提升了约180,000倍。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测，该纯化组分仅呈现单一蛋白条带，其迁移率对应分子量为15 kD，且可回收该条带对应的酶活性。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析，该酶的推导分子量为13,900 D。研究人员测定得到一段含53个氨基酸残基的N端序列，并将其与其他序列进行比对，结果显示其与部分水稻（Oryza sativa）表达序列标签（EST）克隆的推导氨基酸序列具有62%的序列同一性。该纯化酶的最适pH为碱性，且催化活性依赖钙离子（Ca²+）。其对热、酸性环境及有机溶剂具有异常优异的稳定性，但对二硫键还原剂敏感。该酶为典型的PLA₂，既无法水解磷脂酰胆碱的sn-1位点，也对溶血磷脂酰胆碱或二酰甘油无任何催化活性。生化数据与氨基酸序列比对结果均表明，该酶与特征明确的动物分泌型PLA₂家族具有亲缘关系；据我们所知，这是首个被报道的该类型植物源PLA₂酶。