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The Biosynthesis of Apo- and Metalloalkaline Phosphatases of Escherichia coli

From the ¹ From the Department of Biochemistry, Yale University, New Haven, Connecticut 06510

A system for detecting the effects of metal ions, repressors, and mutations on the biosynthesis in vivo of alkaline phosphatase of Escherichia coli is described. If E. coli constitutive for alkaline phosphatase are grown in a medium containing ⁶⁵Zn and are subject to osmotic shock, about 50% of the zinc taken up by the cells is released into the medium. Chromatography of this medium shows that most of the ⁶⁵Zn is associated with alkaline phosphatase. The ratio of specific radioactivity to enzyme shows 2 to 3 g atoms of zinc incorporated per mole of alkaline phosphatase under these conditions. This biosynthetically incorporated zinc is not exchangeable with exogenous zinc added to the medium after osmotic shock. In the case of the wild type organisms, phosphate suppresses but does not abolish incorporation of ⁶⁵Zn into the cell region accessible to osmotic shock. Chromatography shows no ⁶⁵Zn alkaline phosphatase, but does show the presence of another zinc protein unaffected by the presence of phosphate. E. coli mutants, U9 and U47, which produce defective alkaline phosphatases do not incorporate ⁶⁵Zn normally. Under zinc-free conditions (<10^-8 m Zn (II)) E. coli grows normally and synthesizes a zinc-free inactive apoenzyme. The highly purified biosynthesized apoenzyme can be completely reactivated by zinc. Optical rotatory dispersion, circular dichroism, and sedimentation velocity centrifugation reveal that the conformation and quaternary structure of this product do not differ significantly from the enzyme synthesized in the presence of Zn(II). In the presence of Co(II) and Cd(II), the organism can be forced to synthesize alkaline phosphatases containing these metal ions. If equal concentrations of Zn(II) are present, however, the enzyme is synthesized containing only Zn(II).

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				M. F. Hoylaerts, T. Manes, and J. L. Millan Mammalian Alkaline Phosphatases Are Allosteric Enzymes J. Biol. Chem., September 5, 1997; 272(36): 22781 - 22787. [Abstract] [Full Text] [PDF]