Hawrylak K, Stinson R A
Department of Pathology, University of Alberta, Edmonton, Canada.
J Biol Chem. 1988 Oct 5;263(28):14368-73.
When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.
当用从蜡状芽孢杆菌获得的磷脂酰肌醇（PI）磷脂酶C、或用蛋白酶无花果蛋白酶和菠萝蛋白酶处理膜结合的人肝碱性磷酸酶时，释放出的酶是二聚体。在pH 7.6下用丁醇提取质膜产生一种水溶性的聚集形式，PI磷脂酶C也能将其转化为二聚体。当用非离子去污剂（Nonidet P - 40）溶解膜结合酶时，其分子量为四聚体；同样，它也能被PI磷脂酶C或蛋白酶转化为二聚体。在pH 6.6下对全肝组织进行丁醇提取并随后纯化，在非变性条件下电泳得到一种二聚体酶，而在pH 7.6或更高pH值下进行丁醇提取并随后通过免疫亲和层析纯化得到一种天然分子量是二聚体形式两倍的酶。这种高分子量形式在十二烷基硫酸钠变性条件下电泳时显示出一条考马斯亮蓝染色带（分子量 = 83,000），其PI磷脂酶C裂解产物也是如此；这个分子量与在pH 6.6下用丁醇提取从全肝纯化得到的酶相同。这些结果强烈暗示存在一种丁醇激活的内源性酶活性（可能是一种磷脂酶），它在酸性pH下具有最佳活性。在提取和纯化过程中通过维持碱性pH来抑制这种活性会产生一种四聚体酶。碱性磷酸酶，无论是通过对完整质膜进行PI磷脂酶C或蛋白酶处理释放出来的，还是以二聚体形式纯化得到的，都不会吸附到疏水介质上。在pH 7.6下用去污剂或丁醇溶解从质膜释放的碱性磷酸酶，经PI磷脂酶C处理后产生一种不吸附到疏水介质上的二聚体酶，而未处理的制剂则会吸附。这种吸附活性很容易被去污剂释放。同样，在pH 7.6下用丁醇提取从质膜释放的碱性磷酸酶会整合到磷脂酰胆碱脂质体中，而通过PI磷脂酶C从膜释放的酶则不会整合。在pH 6.6下从全肝组织的丁醇提取物中纯化得到的二聚体酶也不会整合。我们得出结论，PI磷脂酶C通过去除磷脂酰肌醇的1,2 - 二酰基甘油部分将碱性磷酸酶的疏水四聚体转化为亲水二聚体。基于这些以及其他人的发现，我们设计了一个碱性磷酸酶转化为其各种形式的模型。
