|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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.