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Regulation of glutamine synthetase:IV. Reversible dissociation and inactivation of glutamine synthetase from Escherichia coli by the concerted action of EDTA and urea

Archives of Biochemistry and Biophysics
Publication Date
DOI: 10.1016/0003-9861(67)90137-3
  • Biology


Abstract Factors influencing subunit association in the glutamine synthetase from Escherichia coli have been studied by means of sucrose gradient sedimentation, disc gel electrophoresis, and light-scattering techniques. The native enzyme is not dissociated by treatments with 0.01 m EDTA, 1.0 m urea, mercaptan, or alkaline pH when tested individually; however, 0.01 m EDTA in combination with 1.0 m urea or alkaline pH will cause dissociation of the enzyme to subunits of about 1 12 the molecular weight of the native enzyme. When the dissociated enzyme is sedimented in a sucrose density gradient containing a mixture of 0.01 m EDTA and 1.0 m urea throughout or is subjected to disc gel electrophoresis in a buffer containing 0.01 m EDTA and 1.0 m urea, the protein migrates as a homogeneous band corresponding to a substance of low molecular weight as expected of the basic subunit species. However, when the dissociated enzyme, obtained by prior treatment with 0.01 m EDTA and 1.0 m urea, is sedimented on a sucrose gradient not containing EDTA and urea, or is subjected to disc gel electrophoresis in a buffer not containing these reagents, the enzyme migrates as a polydispersed system containing 7 or more discrete molecular forms of aggregates that vary in sizes, as would be expected of derivatives containing 1–12 or more subunits. A similar polydispersed system is obtained by treatment of the enzyme with 1.0 m urea plus 2-mercaptoethanol. Disaggregation of the enzyme to basic subunits in the presence of 1.0 m urea and 0.01 m EDTA at pH 8.0 is accompanied by a complete loss in catalytic activity. Reaggregation of the dissociated subunits with a transient restoration of catalytic activity is achieved by the addition of Mn ++ and adjustment of the pH to 7.0.

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