Nègre-Aminou P, Nemenoff R A, Wood M R, de la Houssaye B A, Pfenninger K H
Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver 80262, USA.
J Neurochem. 1996 Dec;67(6):2599-608. doi: 10.1046/j.1471-4159.1996.67062599.x.
Nerve growth cones isolated from fetal rat brain exhibit in their cytosol a robust level of phospholipase A2 activity hydrolyzing phosphatidylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatidylcholine (PC). Western blot analysis with an antibody to the well-characterized cytosolic phospholipase A2 (mol wt 85,000) reveals only trace amounts of this PC- and PE-selective enzyme in growth cones. By gel filtration on Superose 12, growth cone phospholipase A2 activity elutes essentially as two peaks of high molecular mass, at approximately 65 kDa and at well over 100 kDa. Anion exchange chromatography completely separates a PI-selective from a PE-selective activity, indicating the presence of two different, apparently monoselective phospholipase A2 species. The PI-selective enzyme, the predominant phospholipase A2 activity in whole growth cones, is enriched greatly in these structures relative to their parent fractions from fetal brain. This phospholipase A2 is resistant to reducing agents and is found in the cytosol as well as membrane-associated in the presence of Ca2+. However, its catalytic activity is Ca(2+)-independent regardless of whether the enzyme is associated with pure substrate or mixed-lipid growth cone vesicles. The PE-selective phospholipase A2 in growth cones was studied in less detail but shares with the PI-selective enzyme several properties, including intracellular localization, the existence of cytosolic and membrane-associated forms, and Ca2+ independence. Our data indicate growth cones contain two high-molecular-weight forms of phospholipase A2 that share many properties with known, Ca(2+)-independent cytosolic phospholipase A2 species but that appear to be monoselective for PI and PE, respectively. In particular, the PI-selective enzyme may represent a new member of the growing family of cytoplasmic phospholipases A2. The enrichment of the PI-selective phospholipase A2 in growth cones suggests it plays a major role in the regulation of growth cone function.
从胎鼠脑部分离出的神经生长锥，其胞质溶胶中表现出强大的磷脂酶A2活性，可水解磷脂酰肌醇（PI）和磷脂酰乙醇胺（PE），但不能水解磷脂酰胆碱（PC）。用针对已充分表征的胞质磷脂酶A2（分子量85,000）的抗体进行蛋白质免疫印迹分析，结果显示在生长锥中仅存在痕量的这种对PC和PE具有选择性的酶。通过在Superose 12上进行凝胶过滤，生长锥磷脂酶A2活性基本上以两个高分子量峰的形式洗脱，分别约为65 kDa和远超过100 kDa。阴离子交换色谱法完全分离了对PI具有选择性的活性和对PE具有选择性的活性，表明存在两种不同的、显然具有单选择性的磷脂酶A2种类。对PI具有选择性的酶是整个生长锥中主要的磷脂酶A2活性，相对于其来自胎脑的母级分而言，在这些结构中大量富集。这种磷脂酶A2对还原剂具有抗性，在存在Ca2+的情况下，它既存在于胞质溶胶中，也与膜相关。然而，无论该酶是与纯底物还是混合脂质生长锥囊泡相关联，其催化活性都不依赖于Ca(2+)。对生长锥中对PE具有选择性的磷脂酶A2的研究较少，但与对PI具有选择性的酶具有若干共同特性，包括细胞内定位、存在胞质和膜相关形式以及不依赖于Ca2+。我们的数据表明，生长锥含有两种高分子量形式的磷脂酶A2，它们与已知的不依赖于Ca(2+)的胞质磷脂酶A2种类具有许多共同特性，但似乎分别对PI和PE具有单选择性。特别是，对PI具有选择性的酶可能代表了不断增加的胞质磷脂酶A2家族中的一个新成员。生长锥中对PI具有选择性的磷脂酶A2的富集表明它在生长锥功能的调节中起主要作用。
