Ghosh B K, Wouters J T, Lampen J O
J Bacteriol. 1971 Nov;108(2):928-37. doi: 10.1128/jb.108.2.928-937.1971.
Sites of alkaline phosphatase activity have been located by an electron microscopic histochemical (Gomori) technique in vegetative cells of a repressible strain SB15 of Bacillus subtilis, derepressed and repressed by inorganic phosphate, and in a mutant SB1004 which forms alkaline phosphatase in a medium high in phosphate. The sites of enzyme activity were revealed as discrete, dense, and largely spherical bodies of varying sizes (20 to 150 nm). Cells of both repressible and repression-resistant strains acted on a wide variety of phosphate esters (p-nitrophenylphosphate, beta-glycerophosphate, adenosine-5'-phosphate, glucose-6-phosphate, glucose-l-phosphate, adenosine triphosphate, and sodium pyrophosphate) to produce inorganic phosphorus under conditions of alkaline phosphatase assay [0.05 m tris(hydroxymethyl)aminomethane buffer (pH 8.4) containing 2 mm MgCl(2)]. The purified alkaline phosphatase also acted on all these esters, although much less effectively on adenosine triphosphate and sodium pyrophosphate than did the cells. Comparison of the relative utilization of the various substrates by repressed and derepressed cells and purified enzyme suggested the presence of multiple enzymes in the cells. Thus, the cytochemical method of trapping the newly generated inorganic phosphorus determines the location of an alkaline phosphatase of broad substrate profile, and in addition locates the sites of other enzymes generating inorganic phosphorus under identical conditions of assay. It is intriguing that all of these enzymes usually exist in a few clusters attached to the peripheral plasma membrane. In addition to this predominant location, there were a few sites of enzyme activity in the cytoplasm unattached to any discernible structure, and also in the cell wall of the repression-resistant and of the derepressed, repressible strains.
已通过电子显微镜组织化学（Gomori）技术，在枯草芽孢杆菌的可阻遏菌株SB15的营养细胞中定位了碱性磷酸酶活性位点，该菌株在无机磷酸盐作用下发生去阻遏和阻遏，还在突变体SB1004中进行了定位，该突变体在高磷酸盐培养基中形成碱性磷酸酶。酶活性位点显示为大小各异（20至150纳米）的离散、致密且大多呈球形的物体。可阻遏菌株和抗阻遏菌株的细胞在碱性磷酸酶测定条件下[含2 mM MgCl₂的0.05 M三（羟甲基）氨基甲烷缓冲液（pH 8.4）]作用于多种磷酸酯（对硝基苯磷酸酯、β -甘油磷酸酯、腺苷 - 5'-磷酸、葡萄糖 - 6 -磷酸、葡萄糖 - 1 -磷酸、三磷酸腺苷和焦磷酸钠）以产生无机磷。纯化的碱性磷酸酶也作用于所有这些酯类，尽管其对三磷酸腺苷和焦磷酸钠的作用效果远不如细胞。对阻遏和去阻遏细胞以及纯化酶对各种底物的相对利用情况进行比较，表明细胞中存在多种酶。因此，捕获新生成无机磷的细胞化学方法确定了具有广泛底物谱的碱性磷酸酶的位置，此外还确定了在相同测定条件下产生无机磷的其他酶的位点。有趣的是，所有这些酶通常存在于附着于外周质膜的少数簇中。除了这个主要位置外，在细胞质中还有一些未附着于任何可识别结构的酶活性位点，以及在抗阻遏菌株和去阻遏的可阻遏菌株的细胞壁中也有酶活性位点。
