de Carvalho M S, McCormack F X, Leslie C C
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
Arch Biochem Biophys. 1993 Nov 1;306(2):534-40. doi: 10.1006/abbi.1993.1549.
A human, 85-kDa arachidonoyl-specific, cytosolic phospholipase A2 was expressed using the baculovirus-insect cell expression system. Expression resulted in the production of an active protein which consisted of approximately 3% of the total protein in the host Spodoptera frugiperda (Sf9) cells at 67 h after infection. The phospholipase A2 was purified to apparent homogeneity and exhibited calcium-dependent phospholipase A2 activity with a specific activity of 8 mumol/min/mg protein, as well as calcium-independent lysophospholipase activity with a specific activity of 17 mumol/min/mg protein. The phospholipase A2 was expressed as a phosphoprotein and was primarily phosphorylated on serine residues. Phosphatase treatment of the recombinant phospholipase A2 resulted in dephosphorylation of the enzyme and a 63% decrease in phospholipase A2 activity. This decrease in activity is similar in magnitude to the decrease in activity observed with phosphatase-treated phospholipase A2 from stimulated mammalian cells. These data demonstrate that the 85-kDa phospholipase A2 is expressed as an activated phosphoprotein in Sf9 cells.
利用杆状病毒-昆虫细胞表达系统表达了一种分子量为85 kDa的人源花生四烯酰特异性胞质磷脂酶A2。表达产生了一种活性蛋白，在感染后67小时，该蛋白占宿主草地贪夜蛾（Sf9）细胞总蛋白的约3%。磷脂酶A2被纯化至表观均一，表现出钙依赖性磷脂酶A2活性，比活性为8 μmol/min/mg蛋白，以及钙非依赖性溶血磷脂酶活性，比活性为17 μmol/min/mg蛋白。磷脂酶A2以磷蛋白形式表达，主要在丝氨酸残基上磷酸化。对重组磷脂酶A2进行磷酸酶处理导致该酶去磷酸化，磷脂酶A2活性降低63%。这种活性降低的幅度与用磷酸酶处理受刺激哺乳动物细胞中的磷脂酶A2后观察到的活性降低幅度相似。这些数据表明，85 kDa的磷脂酶A2在Sf9细胞中以活化的磷蛋白形式表达。
