Witt W, Schweingruber M E, Mertsching A
Biochim Biophys Acta. 1984 Aug 15;795(1):108-16. doi: 10.1016/0005-2760(84)90110-3.
Two forms of phospholipase B could be solubilized from the plasma membrane of Saccharomyces cerevisiae, separated by gel filtration with Sephacryl S-300 and identified by SDS-polyacrylamide gel electrophoresis as glycoproteins of the apparent molecular weights of about 220 000 (phospholipase B1) and 145 000 (phospholipase B2). The enzymes are very similar in respect to their catalytic properties. Both forms converted lysophosphatidylcholine to diacylphosphatidylcholine and unesterified fatty acids. The carbohydrate content of the glycoproteins could be reduced by treatment with endoglycosidase H and HF. By incubation of phospholipase B1 and phospholipase B2 with endoglycosidase H from Streptomyces griseus, one main protein with an apparent Mr of 67 000 and the same residual carbohydrate content was obtained. Treatment with HF reduced phospholipase B1 and phospholipase B2 to proteins with an apparent Mr of 52 000 and 67 000, respectively. These results could indicate that the two forms are similar in respect to their protein moieties. An antiserum raised in mice against phospholipase B2 showed no crossreactivity with phospholipase B1 as detected by immunoblot analysis. The reactivity of phospholipase B2 was diminished or abolished by progressive removal of carbohydrate. These results were taken as indications for differences in the carbohydrate component of the two enzyme forms.
从酿酒酵母的质膜中可溶解出两种形式的磷脂酶B，它们通过用Sephacryl S - 300进行凝胶过滤分离，并通过SDS - 聚丙烯酰胺凝胶电泳鉴定为表观分子量约为220 000（磷脂酶B1）和145 000（磷脂酶B2）的糖蛋白。这两种酶在催化特性方面非常相似。两种形式都能将溶血磷脂酰胆碱转化为二酰基磷脂酰胆碱和未酯化的脂肪酸。用内切糖苷酶H和HF处理可降低糖蛋白的碳水化合物含量。将磷脂酶B1和磷脂酶B2与来自灰色链霉菌的内切糖苷酶H一起孵育，可得到一种表观分子量为67 000且具有相同残留碳水化合物含量的主要蛋白质。用HF处理分别将磷脂酶B1和磷脂酶B2降解为表观分子量为52 000和67 000的蛋白质。这些结果可能表明这两种形式在蛋白质部分方面相似。通过免疫印迹分析检测，在小鼠中产生的针对磷脂酶B2的抗血清与磷脂酶B1没有交叉反应。随着碳水化合物的逐步去除，磷脂酶B2的反应性降低或消失。这些结果被视为两种酶形式在碳水化合物成分上存在差异的迹象。
