Title: REGULATION OF GLUTAMINE SYN THETASE , VIII. A TP: GLUTAMINE SYNTHIETASE ADENYLYLTRANSFERASE, AN ENZYME THA T CA TALYZES ALTERA TIONS IN THE REGULA TORY PROPERTIES OF GLUTA MINE SYNTHYETASE
Abstract: Elscherichia coti produces two distiniet forrmis of synthetase, depenldinig on the manner in which the organism is grown;1' 2 these forms have been designated synthetases I and HI for convenience. A preparation containing mostly synthetase I was obtained from log-phase cells grown on glucose and NH4C1, and a preparation containing mostly synthetase II from stationary-phase cells grown on glycerol and glutamate.3 The two enzymes are identical in amino acid composition and in sedimentation behavior,4 but synthetase II contains covalently bound AMP which synthetase I lacks.5 This paper describes an (ATP: synthetase adenylyltransferase) derived from E. coli W that catalyzes the conversion of synthetase I to synthetase II in the presence of ATP and Mg++. During this conversiorn the labeled moiety of C14-ATP or a-P32-ATP is converted into a proteinbound form. The was partially purified by a procedure identical to that used by Mecke et at. for the isolation of a glutamine synthetase-inactivating enzyme from E. coli B,6 and it is possible that the adenylyltransferase is analogous to, if not identical with, their enzyme. However, contrary to their results, the biosynthetic activity of the synthetase from E. coli W is not completely lost as a result of the adenylylation reaction, but its specificity for divalent cation activation is greatly altered. These and other properties of the adenylylation reaction and its relationship to the reaction described by Mecke et al.6 are described. Materials and Methods.-Materials: a-P12-ATP and 8-C'4-ATP were obtained from Schwartz BioResearch, Inc., Orangeburg, New York. Preparations of synthetases I and II were obtained by the procedure of Woolfolk et al.7 from E. coli W, grown as previously described.2 The adernylyltransferase was prepared from E. coli W by the procedure which Mecke et al. used in preparing their E. coli B inactivating enzyme.6 The E. coli W cells used for this purpose were grown on glycerol and glutamate in the samne manner used for the production of synthetase II.2 Enzyme assays: Glutamine synthetase was assayed by three methods: (a) the MIg++ biosynthetic assay which involves measuring the release of phosphate from ATP in the presence of glutamate, ammonia, and 50 mM MgCl2 ;7 (b) the Mn++ biosynthetic assay which is the same as (a), except that 5 mM MnCl2 is substituted for the M9gC2; and (C) the y-glutamyl transfer assay which was previously described.2 Radioactivity: Protein-bound radioactivity was determined by precipitation of the radioactive proteini on filter-paper disks as described by Mans anld Novelli,8 excep-t thcat the wCashes with hot trichloracetic acid and ohot ethanol-ether were 011 iittetd.
Publication Year: 2016
Publication Date: 2016-01-01
Language: en
Type: article
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