Hoener M C, Stieger S, Brodbeck U
Abteilung Neurobiochemie, Universität Bern, Switzerland.
Eur J Biochem. 1990 Jul 5;190(3):593-601. doi: 10.1111/j.1432-1033.1990.tb15614.x.
In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.
近年来，越来越多的蛋白质被证明通过共价连接的磷脂酰肌醇聚糖残基锚定在膜上。在哺乳动物细胞中，对于可能在磷脂酰肌醇聚糖锚定蛋白代谢中起作用的磷脂酰肌醇聚糖特异性磷脂酶了解甚少。为了鉴定磷脂酰肌醇聚糖特异性磷脂酶，开发了一种快速灵敏的检测方法，以乙酰胆碱酯酶的磷脂酰肌醇聚糖锚定两亲性膜形式为底物。通过在Triton X-114中分离后水相中可溶性亲水性乙酰胆碱酯酶的增加来监测产物形成的速率。通过该检测方法，我们确定牛脑中存在一种磷脂酰肌醇聚糖特异性磷脂酶。这种酶是可溶的，可以通过加热步骤、随后在DEAE-纤维素上进行色谱分离以及在Sepharose CL-6B上进行凝胶过滤进行部分纯化。磷脂酰肌醇聚糖特异性磷脂酶对底物的磷脂酰肌醇聚糖锚具有高亲和力（Km = 52 nM），并且不降解磷脂酰胆碱或磷脂酰肌醇。用3-三氟甲基-3-（间-[125I]碘苯基）重氮甲烷[（125I]TID）对底物的锚进行疏水标记导致裂解速率显著降低，并且锚的葡糖胺残基氨基的甲基化使裂解速率降至零。使用[125I]TID标记的底物，鉴定出磷脂酰甘油磷酸二酯为第二种产物，表明磷脂酰肌醇聚糖特异性磷脂酶的裂解特异性是磷脂酶D的特异性。磷脂酰肌醇聚糖特异性磷脂酶D受到汞剂、邻菲罗啉和乙二醇双乙醚四乙酸（EGTA）的抑制。它受到微摩尔浓度的Ca2+刺激，表明磷脂酰肌醇聚糖磷脂酶D是一种受Ca2+调节的酶。
