Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta.
Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta.
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发表时间：
1994-09
影响因子：
6.5
通讯作者：
E. Arrese;Michael A. Wells
中科院分区：
文献类型：
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作者：
E. Arrese;Michael A. Wells
一种三酰甘油脂肪酶，可能是第一种参与从昆虫脂肪体中动员脂肪的酶，已经从六分扁豆的脂肪体中纯化成均一种。纯化过程包括聚乙二醇层析、DEAE-纤维素、苯基琼脂糖层析、Q-琼脂糖层析和羟基磷灰石层析。最终产物经SDS-PAGE分析，M(R)=76000，比原匀浆纯化近8000倍，产率约11%。该酶催化三、二和单油酰甘油的水解，但对三或二油酰甘油表现出最高的亲和力。因此，在初始反应条件下，三油酰甘油的最终产物为：游离脂肪酸(66%)、sn-2-单油酰甘油(24%)、sn-1，2(2，3)-二油酰甘油(7%)和甘油(3%)。脂肪体脂酶对三油酰甘油的sn-1或sn-3位没有立体选择性，而对酰基甘油的初级酯键有较强的选择性。该酶的最适pH为7.9，可被氟磷酸二异丙酯、三磷酸腺苷、二磷酸腺苷、镁和氟化钠抑制。该酶有较强的聚集倾向，但在高浓度甘油的洗涤剂溶液中稳定。多肽被来自牛心的cAMP依赖的蛋白激酶磷酸化；然而，磷酸化不会引起该酶的激活。提示该脂肪体脂肪酶可能类似于脊椎动物脂肪组织的激素敏感型脂肪酶。
A triacylglycerol lipase, presumably the first enzyme involved in the mobilization of lipid from the insect fat body, has been purified to homogeneity from the fat body of Manduca sexta. The purification procedure involved polyethyleneglycol precipitation, and chromatography on DEAE-cellulose, phenyl-Sepharose, Q-Sepharose and hydroxylapatite. The final product, a protein with an M(r) = 76,000 by SDS-PAGE, was purified nearly 8000-fold from the original homogenate in a yield of about 11%. The enzyme catalyzed the hydrolysis of tri-, di-, and mono-oleoylglycerols, but showed highest affinity for tri- or dioleoylglycerol. Thus, under initial reaction conditions, the end products of trioleoylglycerol hydrolysis were: free fatty acids (66%), sn-2-monooleoylglycerol (24%), sn-1,2(2,3)-dioleoylglycerol (7%), and glycerol (3%). The fat body lipase exhibited a preference for hydrolyzing the primary ester bonds of acylglycerols, and did not show stereoselectivity toward either the sn-1 or sn-3 position of trioleoylglycerol. The enzyme had a pH optimum of 7.9, and was inhibited by diisopropylfluorophosphate, ATP, ADP, Mg2+, and NaF. The enzyme showed a strong tendency to aggregate, but was stable in detergent solutions at high concentration of glycerol. The polypeptide was phosphorylated by the cAMP-dependent protein kinase from bovine heart; however, phosphorylation did not cause activation of the enzyme. It is suggested that this fat body lipase could be analogous to the "hormone-sensitive lipase" of vertebrate adipose tissue.