Derman A I, Beckwith J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Bacteriol. 1995 Jul;177(13):3764-70. doi: 10.1128/jb.177.13.3764-3770.1995.
Alkaline phosphatase is normally localized to the periplasm of Escherichia coli and is unable to fold into its native conformation if retained in the cytoplasm of growing cells. The alkaline phosphatase activity of E. coli expressing a version of the protein without a signal sequence was nonetheless found to increase gradually when the growth of cells was suspended. At least 30% of the protein was activated over the course of several hours when freshly grown exponential-phase cells were held on ice. Similar behavior was observed with cells expressing certain other mutant versions of alkaline phosphatase that are retained in the cytoplasm. The activation resulted not from the passage of the alkaline phosphatase into the periplasm but from the slow folding of alkaline phosphatase into its native conformation in the cytoplasm. These findings indicate that the mechanism by which proteins are normally kept reduced in the cytoplasm fails to function if cells are not growing. It was found that the addition of the sulfhydryl-alkylating agent iodoacetamide to cells after growth blocks this activation completely. This treatment can therefore diminish the likelihood of spurious enzyme activity measurements in studies that make use of alkaline phosphatase fusion proteins.
碱性磷酸酶通常定位于大肠杆菌的周质中，如果保留在生长细胞的细胞质中则无法折叠成其天然构象。然而，当细胞生长暂停时，表达没有信号序列的该蛋白版本的大肠杆菌的碱性磷酸酶活性仍会逐渐增加。当新鲜生长的指数期细胞置于冰上时，至少30%的蛋白在数小时内被激活。在表达保留在细胞质中的碱性磷酸酶的某些其他突变版本的细胞中也观察到了类似的行为。这种激活不是由于碱性磷酸酶进入周质，而是由于碱性磷酸酶在细胞质中缓慢折叠成其天然构象。这些发现表明，如果细胞不生长，蛋白质通常在细胞质中保持还原状态的机制就会失效。研究发现，生长后向细胞中添加巯基烷基化剂碘乙酰胺会完全阻断这种激活。因此，这种处理可以降低在使用碱性磷酸酶融合蛋白的研究中出现假酶活性测量的可能性。
