Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme.

The PII regulatory protein of Escherichia coli glutamine synthetase exists in two interconvertible forms: a uridylylated form (PIID) which promotes the deadenylylation of glutamine synthetase and an unmodified form (PIIA) which promotes the adenylylation of glutamine synthetase (Mangum, J.H., Magni, G., and Stadtman, E.R. (1973) Arch. Biochem. Biophys. 158, 514-525). PII has been purified to homogeneity. Its molecular weight is 44,000. The protein is composed of four subunits, each with a molecular weight of approximately 11,000. The subunits are identical as judged by: (a) the homogeneity of the subunits in sodium dodecyl sulfate, 8 M urea, and 6 M guanidine HCl; (b) the minimal molecular weight calculated from the amino acid composition; and (c) the isolation of only two tryptic peptides containing tyrosine (there are 8 tyrosyl residues per 44,000 molecular species). Following iodination of PIIA and PIID with 125I in the presence of chloramine-T, tryptic digestion yields two radioactive peptides from PIIA and only one from PIID. Since a tyrosine with a substituted hydroxyl group cannot be iodinated, this result indicates that 1 tyrosyl residue in each subunit is modified by the covalent attachment of UMP. This conclusion is supported also by the fact that treatment of PIID with snake venom phosphodiesterase results in the release of covalently bound UMP and the stoichiometric appearance of phenolate ion (pH 13) as measured by ultraviolet absorption spectroscopy. The enzyme activities (uridylyl-removing) responsible for removal and (uridylytransferase) responsible for attachment of UMP to PII have been partially purified. These activities co-purify through a variety of procedures, including hydrophobic chromatography, and are stabilized by high ionic strength buffers. Whereas Mn2+ alone supports only uridylyl-removing activity, ATP, alpha-ketoglutarate, and Mg2+ support both uridylyl-removing and uridylyltransferase activities.