Shiomi K A, Kazama A, Shimakura K, Nagashima Y
Department of Food Science and Technology, Tokyo University of Fisheries, Japan.
Toxicon. 1998 Apr;36(4):589-99. doi: 10.1016/s0041-0101(97)00085-8.
Two phospholipases A < inf2 (named AP-PLA2-I and II) were purified from the crown-of-thorns starfish (Acanthaster planci) venom. Both enzymes were confirmed to be PLA2s, based on the results that they showed hemolytic activity only in the presence of phosphatidylcholine (PC) and also released fluorescent fatty acids from PC with labeled fatty acids at the sn-2 position. The enzyme activity of both PLA2s was enhanced by Ca2+ but reduced by Cu2+ and Zn2+. The molecular mass of AP-PLA2-I was estimated to be 28 kDa by gel filtration and 15 kDa by SDS-PAGE, indicating that AP-PLA2-I is a dimer composed of the same subunit. In contrast, AP-PLA2-II was judged to be a monomer with a molecular mass of 12 kDa (gel filtration) or 15 kDa (SDS-PAGE). The amino acid compositions of the two enzymes were comparable to each other; Asx, Glx and Gly were rich in both molecules, while Met, His and Trp were poor. Analyses by a sequencer determined the first 62 amino acid residues for both PLA2s. In the AP-PLA2-I preparation, minor amino acids were additionally found at 17 positions, suggesting the coexistence of another PLA2-component. As compared to the N-terminal sequences of the known PLA2s, both AP-PLA2-I and II were identified as class I enzymes not only because they have Cys-11 and lack Cys-51 but also because they contain the elapid loop in the region 53-61.
从棘冠海星（Acanthaster planci）毒液中纯化出了两种磷脂酶A2（分别命名为AP-PLA2-I和II）。基于它们仅在磷脂酰胆碱（PC）存在时表现出溶血活性，并且还能从sn-2位带有标记脂肪酸的PC中释放出荧光脂肪酸这一结果，证实这两种酶均为磷脂酶A2。两种磷脂酶A2的酶活性均因Ca2+而增强，但因Cu2+和Zn2+而降低。通过凝胶过滤法估计AP-PLA2-I的分子量为28 kDa，通过SDS-PAGE法估计为15 kDa，这表明AP-PLA2-I是由相同亚基组成的二聚体。相比之下，AP-PLA2-II被判定为单体，分子量为12 kDa（凝胶过滤法）或15 kDa（SDS-PAGE法）。这两种酶的氨基酸组成彼此相当；Asx、Glx和Gly在两个分子中都很丰富，而Met、His和Trp则较少。通过测序仪分析确定了两种磷脂酶A2的前62个氨基酸残基。在AP-PLA2-I制剂中，在17个位置还额外发现了少量氨基酸，这表明还存在另一种磷脂酶A2成分。与已知磷脂酶A2的N端序列相比，AP-PLA2-I和II均被鉴定为I类酶，这不仅是因为它们具有Cys-11且缺少Cys-51，还因为它们在53-61区域含有眼镜蛇毒环。
