Hatch G M, Vance D E, Wilton D C
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochem J. 1993 Jul 1;293 ( Pt 1)(Pt 1):143-50. doi: 10.1042/bj2930143.
A novel fluorescence assay for phospholipase A2 [Wilton (1990) Biochem. J. 266, 435-439] has been used to study the Group-II rat liver mitochondrial enzyme, and a number of novel properties of this enzyme were identified. (1) The enzyme activity was located in the liver macrophages (Kupffer cells) while negligible activity was associated with hepatocytes. (2) Although subcellular fractionation of whole liver confirmed the predominantly mitochondrial location of this enzyme activity, the analysis of the hepatocyte-free Kupffer-cell-enriched fraction revealed a different enzyme distribution, with the majority of activity being associated with the microsomal membrane fraction. (3) Bacterial endotoxin has been previously shown to be scavenged by Kupffer cells in rats. Treatment of rats with bacterial lipopolysaccharide (endotoxin) resulted in a dramatic time- and dose-dependent increase in liver phospholipase A2 activity. (4) It is known that injection of endotoxin into rodents results in elevated serum phospholipase A2 activity, while a similar phenomenon is seen in the condition of septic shock in man. The source of this serum enzyme was unknown. In this study perfusion of livers from rats pretreated with lipopolysaccharide with physiological saline demonstrated a 6-fold increase in phospholipase A2 activity in the perfusate compared with sham-treated controls, with only minor release of hepatic lipase. (5) Western-blot analysis confirmed an increased release of this Group-II phospholipase A2 into the perfusate of lipopolysaccharide-treated rats compared with sham-treated controls. These results suggest that liver Kupffer cells are a major source of the endotoxin-induced serum Group-II phospholipase A2 activity associated with bacterial infection and trauma.
一种用于磷脂酶A2的新型荧光测定法[威尔顿（1990年），《生物化学杂志》266卷，435 - 439页]已被用于研究II组大鼠肝脏线粒体酶，并鉴定出该酶的一些新特性。（1）酶活性位于肝脏巨噬细胞（库普弗细胞）中，而与肝细胞相关的活性可忽略不计。（2）虽然对整个肝脏进行亚细胞分级分离证实了该酶活性主要位于线粒体中，但对不含肝细胞的富含库普弗细胞的分级分离物的分析揭示了不同的酶分布，大部分活性与微粒体膜分级分离物相关。（3）先前已表明细菌内毒素可被大鼠的库普弗细胞清除。用细菌脂多糖（内毒素）处理大鼠导致肝脏磷脂酶A2活性出现显著的时间和剂量依赖性增加。（4）已知向啮齿动物注射内毒素会导致血清磷脂酶A2活性升高，而在人类脓毒症休克的情况下也会出现类似现象。这种血清酶的来源尚不清楚。在本研究中，用生理盐水灌注经脂多糖预处理的大鼠的肝脏，结果表明与假处理对照组相比，灌注液中的磷脂酶A2活性增加了6倍，同时肝脂肪酶仅有少量释放。（5）蛋白质免疫印迹分析证实，与假处理对照组相比，脂多糖处理的大鼠的灌注液中这种II组磷脂酶A2的释放增加。这些结果表明，肝脏库普弗细胞是与细菌感染和创伤相关的内毒素诱导的血清II组磷脂酶A2活性的主要来源。
