Earlier studies showed that two protein A complex enzyme system that catalyzes adenylylation and deadenylylation of Escherichia coli glutamine synthetase (EC 6.3.1.2) controls its activity and its response to metabolic effectors (1-7). A protein of molecular weight about 130,000, designated PI (5), catalyzes both adenylylation (reaction 1) and deadenylylation (reaction 2). glutamine synthetase + ATP -AMP-synthetase + PP1 (1) AMP-synthetase + Pi --ADP + glutamine synthetase (2) A second protein of molecular weight 50,000 designated PI,, stimulates (3, 5) reaction 2. As originally studied (3, 5, 7), PI, had no effect on reaction 1.We report here that PI, can exist in two forms. One, designated Pi,-DA, resembles the form previously described (5); it stimulates Pi-catalyzed deadenylylation (reaction 2) but has little effect on the adenylylation (reaction 1). The other form, designated P1i-AT, markedly stimulates reaction 1, but has little or no effect on reaction 2. In the presence of 2-oxoglutarate, UTP, and ATP, an enzyme present in preparations of P1 catalyzes the conversion of Pi,-AT to Pi,-DA. ['y-32P]UTP (3.5 Ci/mmol) was obtained from International Chemical and Nuclear Corp. The purity of all isotopic compounds was confirmed by thin-layer chromatography. Dithiothreitol (DTT) and 2-oxo glutarate were from Calbiochem. L-glutamine and iglutamate were from Sigma. All nucleotides were from P-L Biochemicals; Sephadex G-100 was from Pharmacia.Enzymes. Glutamine synthetase was purified by a zinc precipitation method (R. Miller and E. R. Stadtman, unpublished procedure) from E. coli W cells grown on a medium containing 75 mM NH4Cl and 0.67 M glycerol and harvested after several hours in the stationary phase. This enzyme contained an average of 0.8 equivalent of AMP per mole (E6-.8).The [14C]adenylylated glutamine synthetase, which served as a substrate for all the deadenylylation experiments, was prepared enzymatically (8) by adenylylation of Eo-with ['4C]ATP. The glutamine synthetase so prepared contained 11.4 equivalents of AMP per mole (E-i.i), with a specific activity of 20 cpm/pmol of adenylylated subunits.Partially purified preparations of PI and PI, were obtained (5) from E. coli W cells grown on a medium containing 0.67 M glycerol and either 38 or 76 mM NH4Cl. The cells were harvested about 12 hr after the onset of stationary phase. Purity of the PI and Pi,-preparations finally obtained was estimated to be about 40 and 70%O, respectively. Both enzymes were stable for several months when stored at -80°C in 20 mM Tris-0.5 MM DTT-0.25 mM Na2MgEDTA (pH 7.5).Enzyme Askat. The assay mixture (0.1 ml) for estimation of deadenylyllting activity (DA-activity) contained 50 mM 2-methylimidazole HCl (pH 7.2), 0.02 mM ATP, 1.0 mM UTP, 15 mM 2-oxoglutarate, 20 mM MgCl2, 5 mM NarMgEDTA, 20 mM K phosphate, 1 mM DTT, 100 ,ug of[I4C]ATP-adenylylated glutamine synthetase (equal to 2.0 nmol of adenylylated subunits, 20 X 101 cpm'nmol) and enzyme. After 30 min at 37°C, 0.15 ml of 6% HC104 was added and the mixtures were cen...