Properties of Ca2+-independent phospholipase A2 and its roles in phospholipid fatty acid remodeling

磷脂酶A2（PLA 2）代表了一个不同的酶家族，并且根据活性对Ca^2+离子的需要可以分为两大类，Ca^2+依赖性和Ca^2+非依赖性PLA 2。本研究的目的是探讨钙非依赖性磷脂酶A2的分子多样性及其在磷脂代谢，特别是脂肪酸重构中的生理意义.大鼠睾丸含有两种形式的钙非依赖性PLA，即我们从大鼠小肠中纯化和克隆的肠平滑肌磷脂酶B/脂肪酶（PL B/LIP）和酸性磷脂偏好性PLA（PLA）。后一种酶占睾丸中可测量的PLA活性的大部分。我们部分纯化了这种酶，并发现PLA活性共洗脱与脂肪酶活性相同的PLA/脂肪酶活性比在每个色谱步骤，这表明一个单一的酶催化这些活动。PLA的底物特异性和表观分子量与PLB/LIP不同，为了对其进行详细的结构和功能研究，我们正在改进纯化策略。由于在睾丸中，以花生四烯酸单酰甘油为中间体的磷脂脂肪酸重构是活跃的，因此明确这些具有脂肪酶活性的聚乳酸在重构系统中的作用具有重要意义.免疫组化显示PLB/LIP存在于精子顶体。我们正在研究其与顶体反应的相关性。3.定点突变和化学修饰研究表明，PLB/LIP属于一个新的脂肪酶家族，在G-D-S-L基序中有一个活性位点Ser-404，并鉴定了催化必需的His和Asp.我们建立了PLB/LIP的杆状病毒表达系统和一种快速纯化表达酶的两步法。我们现在正试图制备适合于X射线晶体学的单晶。

Phospholipases A2 (PLA2) represent a diverse family of enzymes and can be classified into broad two classes according to requirement of Ca^<2+> ions for activity, Ca^<2+>-dependent and Ca^<2+>-independent PLA2s. The aim of this study is to explore the molecular diversity of Ca^<2+>-independent PLA2s and their physiological significance in phospholipid metabolism, especially fatty acid remodeling.1. Rat testis contained two forms of Ca^<2+>-independent PLAs, phospholipase B/lipase (PLB/LIP) of the intestinal tyoe, which we purified and cloned from rat small intestine, and acidic phospholipid-preferring PLA (PLA). The latter enzyme accounted for the majority of measurable PLA activity in the testus. We partially purified this enzyme, and found that PLA activity coeluted with lipase activities with the same PLA/lipase activity ratios at each chromatographic step, suggesting a single enzyme catalyzes these activities. The substrate specificity and apparent molecular mass of the PLA differed from those of PLB/LIP.For its detailed structural and functional studies, we are now improving purification strategies. Since in the testis phospholipid fatty acid remodeling involving arachidonoyl monoacylglycerol as an intermediate is active, it is important to clarity the roles of these PLA with lipase activity in the remodeling system.2. Immunohistochemistry showed that PLB/LIP is present in acrosomes of sperm. We are investigating its relevance to acrosome reactions.3. Site-directed mutagenesis and chemical modification studies indicated that PLB/LIP belongs to a new lipase family : an active site Ser-404 was located in a motif G-D-S-L.Furthermore, we identified a His and an Asp essential for catalysis.4. We developed an baculovirus expression system for PLB/LIP and a rapid twostep method for purifying the expressed enzyme. We are now trying to prepare its single crystals suitable for x-ray crystallography.