Glutamine synthetase adenylylation in permeabilized cells of Escherichia coli.
Following a freeze-thaw cycle, treatment of Escherichia coli with the nonionic detergent, Lubrol WX, renders the cells permeable to small molecules but not to cytosolic proteins. After such treatment, the permeabilized cell suspensions can be assayed directly by standard procedures both for intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number, n, of adenylylated subunits/dodecameric molecule). Permeabilization of cells from cultures containing an adequate supply of glutamine as the sole nitrogen source led to complete retention of all protein components of the bicyclic cascade that regulates the interconversion of glutamine synthetase between adenylylated and unadenylylated forms; similar treatment of glutamine-starved cells leads to selective inactivation, only, of the uridylyltransferase. When suspended in buffers containing ATP and glutamine, the value of n in permeabilized cells increased to high values (n = 11), whereas in the presence of alpha-ketoglutarate, Pi, and ATP, the value of n decreased to approximately 2.0. Time-dependent changes in n that occur during incubations of permeabilized cells in buffers containing these effectors can be arrested either by sonication at 0-4 degrees C or by the addition of cetyltrimethylammonium bromide (to inactivate adenylyltransferase). It is thus evident that Lubrol-treated cells may be used to investigate the regulation of glutamine synthetase adenylylation in situ.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013696:Temperature; D005723:gamma-Glutamyltransferase / Q000378:metabolism
C017474:Lubrol WX; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D005723:gamma-Glutamyltransferase; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; D005974:Glutamate-Ammonia Ligase