Heller M, Bütikofer P, Brodbeck U
Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
Eur J Biochem. 1994 Sep 15;224(3):823-33. doi: 10.1111/j.1432-1033.1994.00823.x.
It has been suggested previously that small amounts of the mature 115-kDa form of phosphatidylinositol (PtdIns)-glycan-specific phospholipase D from bovine serum may exist as a 47-kDa form which can also be generated in vitro by treatment with proteases. In this study, we investigated the possible proteolytic processing by trypsin of partially purified PtdIns-glycan- specific phospholipase D from bovine serum and found that tryptic digestion caused an apparent activation of the enzyme when assayed in the presence of 0.1% (mass/vol.) Triton X-100. Trypsin cleaved the 115-kDa form of PtdIns-glycan-specific phospholipase D into three major polypeptides with molecular masses of 33, 39, and 47 kDa. Under non-denaturing conditions, the polypeptides remained tightly but noncovalently associated with each other. However, in the presence of 6 M urea, the polypeptides could be separated by anion-exchange chromatography. After renaturation, PtdIns-glycan-specific phospholipase D activity was found to be associated with a 39-kDa fragment. Based on its size and its amino acid sequence, the active-site-containing fragment consisted of approximately 275 residues of the N-terminal region of PtdIns-glycan-specific phospholipase D. The active 39-kDa fragment hydrolyzed the PtdIns-glycan-anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from blood stream trypanosomes. However, this fragment was inactive on membrane-associated acetylcholinesterase and PtdIns.
先前有人提出，牛血清中少量成熟的115 kDa形式的磷脂酰肌醇（PtdIns）-聚糖特异性磷脂酶D可能以47 kDa形式存在，该形式也可在体外通过蛋白酶处理产生。在本研究中，我们研究了胰蛋白酶对部分纯化的牛血清PtdIns-聚糖特异性磷脂酶D的可能蛋白水解加工，发现在0.1%（质量/体积） Triton X-100存在下进行测定时，胰蛋白酶消化导致该酶明显激活。胰蛋白酶将115 kDa形式的PtdIns-聚糖特异性磷脂酶D切割成三种主要多肽，分子量分别为33、39和47 kDa。在非变性条件下，这些多肽彼此紧密但非共价结合。然而，在6 M尿素存在下，这些多肽可通过阴离子交换色谱分离。复性后，发现PtdIns-聚糖特异性磷脂酶D活性与一个39 kDa片段相关。基于其大小和氨基酸序列，含活性位点的片段由PtdIns-聚糖特异性磷脂酶D N端区域的约275个残基组成。活性39 kDa片段水解了来自牛红细胞的可溶性乙酰胆碱酯酶和来自血流锥虫的可变表面糖蛋白的PtdIns-聚糖锚。然而，该片段对膜相关的乙酰胆碱酯酶和PtdIns无活性。
