Heller M, Bieri S, Brodbeck U
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Biochim Biophys Acta. 1992 Aug 24;1109(2):109-16. doi: 10.1016/0005-2736(92)90073-u.
It has been reported that rat liver membranes contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) which may be involved in generation of phosphoinositol-glycan, a putative insulin second messenger (Saltiel, A.R. and Cuatrecasas, P. (1988) Am. J. Physiol. 255, C1-C11). Using GPI-anchored acetylcholinesterase (AChE) from bovine erythrocytes as substrate, we attempted to isolate GPI-PLC from bovine and rat liver membranes. A major part of the GPI-anchor converting activity present in liver could be washed away from the tissue by extraction with detergent-free buffer. Solubilisation of the washed membranes with 0.25% (v/v) Nonidet P-40 and ultracentrifugation resulted in a considerable amount of detergent soluble GPI-anchor converting activity in the supernatant. Anion-exchange chromatography on a Fractogel TSK-DEAE column of detergent-soluble GPI-anchor converting activity revealed two distinct peaks eluting at 50-80 mM and 120-170 mM NaCl, respectively. Using [125I]TID-labelled mf-AChE as substrate, radiolabelled diradylglycerol was obtained with both peak activities. However, when the phosphatase inhibitors NaF and sodium orthovanadate were included in the assay systems, phosphatidic acid was detected in addition to diradylglycerol. Both GPI-anchor converting activities were Ca(2+)-sensitive and inhibited by heavy metal chelating agents. These results suggested the presence of two isoenzymes of GPI-PLD and a phosphatase, rather than a GPI-PLC activity, in liver. Further, it could be shown that the activity in the second peak was identical to GPI-PLD, abundantly present in serum, while the activity contained in the first peak seems to be genuine for liver cells and, thus, apparently represents a novel form of a GPI-PLD which is membrane-associated and distinctly different from the serum enzyme.
据报道，大鼠肝细胞膜含有一种糖基磷脂酰肌醇特异性磷脂酶C（GPI-PLC），它可能参与磷酸肌醇聚糖的生成，磷酸肌醇聚糖是一种假定的胰岛素第二信使（萨尔蒂尔，A.R.和夸特雷卡斯，P.（1988年）《美国生理学杂志》255卷，C1 - C11）。我们以牛红细胞的GPI锚定乙酰胆碱酯酶（AChE）为底物，尝试从牛和大鼠肝细胞膜中分离GPI-PLC。肝脏中存在的大部分GPI锚转换活性可以通过用无洗涤剂缓冲液提取而从组织中洗去。用0.25%（v/v）的Nonidet P - 40溶解洗涤后的膜并进行超速离心，导致上清液中有相当数量的洗涤剂可溶性GPI锚转换活性。对洗涤剂可溶性GPI锚转换活性在Fractogel TSK - DEAE柱上进行阴离子交换色谱分析，发现有两个不同的峰，分别在50 - 80 mM和120 - 170 mM NaCl浓度下洗脱。以[125I]TID标记的mf - AChE为底物，两个峰活性都能产生放射性标记的二酰基甘油。然而，当在测定系统中加入磷酸酶抑制剂氟化钠和原钒酸钠时，除了二酰基甘油外还检测到了磷脂酸。两种GPI锚转换活性都对Ca(2+)敏感，并受到重金属螯合剂的抑制。这些结果表明肝脏中存在两种GPI-PLD同工酶和一种磷酸酶，而不是GPI-PLC活性。此外，可以证明第二个峰中的活性与血清中大量存在的GPI-PLD相同，而第一个峰中的活性似乎是肝细胞特有的，因此，显然代表了一种与膜相关且与血清酶明显不同的新型GPI-PLD。
