Micrococcal nuclease cleavage of nucleotide linked to glutamme synthetase yields phosphotyrosine at the ligation site ( adenylylated glutamine synthetase / micrococcal nuclease ) TODD

The activity of micrococcal nuclease was studied on a novel substrate, denatured adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2], which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP. The products of the digestion were adenosine and O-phosphotyrosylglutamine synthetase. The Km of the macromolecular substrate with the nuclease was 1/40 that of the synthetic substrate, nitrophenyl-pdT, which is commonly used for assay ofthe enzyme. Native adenylylated glutamine synthetase was not deadenosylated by micrococcal nuclease under the conditions that permit rapid deadenosylation of denatured glutamine synthetase. Failure to attack native glutamine synthetase is probably not due to steric factors because the native enzyme is deadenylylated by snake venom phosphodiesterase under identical conditions. The inability of micrococcal nuclease to deadenosylate native glutamine synthetase may be due to the formation of an inactive complex because the native protein inhibited the nuclease activity on the denatured protein. Regulation of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] activity in Escherichia coli involves the covalent attachment of an adenylyl group in phosphodiester linkage to a single tyrosine residue in each Mr 50,000 subunit (1). Both adenylylation and deadenylylation of the enzyme are catalyzed by the same adenylyltransferase (2), whose activity is regulated by the interconversion ofa small regulatory protein PI,, between uridylylated and nonuridylylated states (3). Uridylylation of the PI, protein involves attachment of 5'UMP through a phosphodiester bond to one of two tyrosine residues in each Mr 11,000 subunit ofthe protein (4). Recently, the modification of several other proteins has been shown to involve the nucleotidylylation of tyrosine residues. The RNA genome of polio virus is ligated to a protein tyrosine residue through a phosphodiester bond (5, 6), and DNA is ligated to topoisomerases through a tyrosine residue (7, 8), probably as a reaction intermediate. Characterization of tyrosine as the site of nucleotide ligation can be demonstrated by both enzymatic and chemical hydrolyses. Alkaline pH difference spectroscopy at 293 nm demonstrated that snake venom phosphodiesterase treatment of an adenylylated decapeptide produced a free phenolic hydroxyl (1). Protein linked to 32P-labeled polynucleotide was enzymatically digested with nucleases to yield protein-pUp; partial acid hydrolysis ofthe latter yielded, among several products, phosphotyrosine and tyrosine-pUp, which was further degraded with micrococcal nuclease to tyrosine phosphate and 3'-UMP (5, 6). We demonstrate here that micrococcal nuclease treatment of denatured adenylylated glutamine synthetase yields phosphotyrosyl glutamine synthetase and adenosine. The procedure allows the preparation ofmilligram quantities ofprotein containing a single residue of phosphotyrosine, which can be utilized to study chemical techniques for tyrosine phosphate quantification and protein phosphotyrosine phosphatase activities in cells and tissues. MATERIALS AND METHODS Glutamine synthetase preparations with different adenylylation states were obtained from E. coli cultured under various degrees of nitrogen availability. The enzyme was purified by Zn2+, acetone, and (NH4)2SO4 precipitation steps (9, 10). Adenylylation reactions were carried out with 1 mM [2,8,5'-3H]ATP or [a-32P]ATP (specific activity, 100-500 jiCi/tumol; 1 Ci = 3.7 X 1010 becquerels) in 25 mM Tris HCI buffer (pH 7.6) containing 20 mM Mg2+, 12.7 mM Gln, 0.15 M KCI, 2 mM phosphoenolpyruvate, pyruvate kinase (=5 units/ml), and sufficient adenylyltransferase to complete the reaction in 30 min. The modified protein was separated from the reaction components by chromatography on Sephadex G-25 (fine) in 10 mM imidazole, pH 7.1. Carboxymethylation of glutamine synthetase was carried out by a method similar to that of Shapiro and Stadtman (11). Prior to the addition ofiodoacetate (20 mM), the enzyme was dialyzed against 6 M urea/i mM EDTA/0.2 M Tris HCI, pH 8.0, and then incubated with 10 mM dithiothreitol for 30 min at 37°C. After carboxymethylation at 4°C (2 hr) in the dark, the sample was exhaustively dialyzed against 1.0 mM sodium borate (pH 9.3). The sample was stored at -20°C after removal of an aliquot for protein (12), phosphate (13), and radioactivity measurements. Micrococcal nuclease and bacterial alkaline phosphatase were obtained from P-L Biochemicals. Snake venom phosphodiesterase was obtained from Boehringer Mannheim. The nuclease (1.0-5.0 mg) was dissolved in 1.0 ml of 20 mM Tris HCI, pH 7.6/20% glycerol. Digestions of native and carboxymethylated adenylyl glutamine synthetase were normally carried out at 37°C in 40-50 mM borate, pH 9.3/ 8-10 mM CaC12. Fixed time assays were carried out in 50 X 6 mm culture tubes in 25to 50-,ul volumes. Reactions were stopped by the addition of 100 IlI of 0.1 M HCl containing bovine serum albumin as carrier (5 mg/ml), followed by 500 IlI of 10% trichloroacetic acid. The samples were centrifuged after standing on ice for 15 min, and an aliquot of the supernatant was removed for scintillation counting. Snake venom phosphodiesterase assays of native adenylylated glutamine synthetase were carried out by using a similar technique. Thin-layer chromatography was carried out on silica gel plates (Eastman) developed with n-propanol:H20:0.1 M NH40H, 3:1:0.1 (vol/ vol).