Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis

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Abstract
Polyacrylamide gel electrophoresis was used to examine the regulation and adenylylation states of glutamine synthetases [EC 6.3.1.2] (GS) from Escherichia coli (GSE) and Klebsiella aerogenes [pneumoniae] (GSK). In gels containing sodium dodecyl sulfate (SDS), GSK had a mobility which differed significantly from that of GSE. For both GSK and GSE, adenylylated subunits (GSK-adenosine 5''-monophosphate [AMP] and GSE-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GSK-AMP < GSK < GSE-AMP < GSE. These mobility differences were detected with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels showed that: the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GSK, and strains carrying the glnA10 allele contained no polypeptide having the mobility of GSK or GSK-AMP. Using native polyacrylamide gels, the increased amount of dodecameric GS present in cells grown under N limitation was detected compared with cells grown under conditions of N excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GS nor a GS-like protein in cells carrying the glnA10 allele.