Day D F, Ingram J M
Can J Microbiol. 1975 Jan;21(1):9-16. doi: 10.1139/m75-002.
Alkaline phosphatase (APase) of Pseudomonas aeruginosa exists primarily in the periplasmic region of the cell, i.e., between the cytoplasmic membrane and the outer tripartite layer. The enzyme is also found in the culture filtrate or associated with the outer layer of the cell wall. APase forms a complex with released outer cell wall material, and lipopolysaccharide (LPS) is associated with the complex. Since the enzyme was purified to homogeneity, it became desirable to determine whether complex formation with LPS , or the outer cell wall, affected any properties of the purified phosphatase. The ratio of activities of purified APase with p-nitrophenylphosphate and beta-glycerolphosphate as substrates is about 4:1. The ratio of activities with enzyme complexed with LPS is about 1:1. The energy of activation of sucrose or magnesium released enzyme is 9500 cal/mol whereas the values for purified enzyme plus LPS, purified enzyme, purified enzyme plus phosphatidylethanolamine (PE), and purified enzyme plus LPS plus PE range from 3400 to 8700 cal/mol. These changes occur in the physiological temperature range, 27 to 39C, of this organism. Sucrose-released enzyme in the presence of substrate is inactivated at 47C whereas pure enzyme plus substrate is affected at 41C. The addition of LPS, PE, or a combination of both increases the temperature of inactivation from 45 to 51C. The results suggest that certain properties of the purified enzyme differ from those of the enzyme released from whole cells by either sucrose or magnesium resuspension. The addition of cell wall components such as LPS and PE to purified APase restores these properties. The addition of cell wall components such as LPS and PE to purified APase restores these properties. The evidence suggests that artificial complex formation changes the environment of the enzyme protein such that the environment now resembles that which exists within the whole cell wall.
铜绿假单胞菌的碱性磷酸酶（APase）主要存在于细胞的周质区域，即细胞质膜和外部三层结构之间。该酶也存在于培养滤液中或与细胞壁外层相关联。APase与释放的细胞壁外层物质形成复合物，脂多糖（LPS）与该复合物相关联。自从该酶被纯化至同质后，就有必要确定与LPS或细胞壁外层形成复合物是否会影响纯化磷酸酶的任何特性。以对硝基苯磷酸酯和β-甘油磷酸酯为底物时，纯化APase的活性比约为4:1。与LPS复合的酶的活性比约为1:1。蔗糖或镁释放的酶的活化能为9500卡/摩尔，而纯化酶加LPS、纯化酶、纯化酶加磷脂酰乙醇胺（PE）以及纯化酶加LPS加PE的值在3400至8700卡/摩尔之间。这些变化发生在该生物体27至39℃的生理温度范围内。底物存在时，蔗糖释放的酶在47℃失活，而纯酶加底物在41℃受到影响。添加LPS、PE或两者的组合可使失活温度从45℃提高到51℃。结果表明，纯化酶的某些特性与通过蔗糖或镁重悬从全细胞释放的酶的特性不同。向纯化的APase中添加LPS和PE等细胞壁成分可恢复这些特性。向纯化的APase中添加LPS和PE等细胞壁成分可恢复这些特性。证据表明，人工复合物的形成改变了酶蛋白的环境，使得该环境现在类似于整个细胞壁内存在的环境。
