Stewart Allison, Ghosh Moumita, Spencer Diane M, Leslie Christina C
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
J Biol Chem. 2002 Aug 16;277(33):29526-36. doi: 10.1074/jbc.M204856200. Epub 2002 May 30.
The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.
对胞质磷脂酶A2γ（cPLA2γ）的酶学特性进行了体外研究，并观察了该酶在细胞中表达时的情况。cPLA2γ是85 kDa的IV型cPLA2α（cPLA2α）的一种同工型。在Sf9细胞中表达的cPLA2γ与膜相关。从表达cPLA2γ的[3H]花生四烯酸标记的Sf9细胞中分离的膜可组成性释放[3H]花生四烯酸。膜相关活性受到IV型磷脂酶A2抑制剂甲基花生四烯酰氟磷酸酯的抑制，但不受VI型磷脂酶A2抑制剂（E）-6-（溴亚甲基）-3-（1-萘基）-2H-四氢吡喃-2-酮的有效抑制。cPLA2γ的溶血磷脂酶活性高于磷脂酶A2活性。纯化的His-cPLA2γ不表现出磷脂酶A1活性，但可依次从磷脂酰胆碱的sn-2和sn-1位水解脂肪酸。在HEK293细胞中过表达的cPLA2γ在分离的膜中具有组成性活性，可释放大量油酸、花生四烯酸、棕榈酸和硬脂酸；然而，完整细胞的基础脂肪酸释放并未增加。在cPLA2α缺陷小鼠的肺成纤维细胞中过表达的cPLA2γ可被小鼠血清激活，导致花生四烯酸、油酸和棕榈酸的释放，而过表达cPLA2α主要导致花生四烯酸的释放。
