Isolation and characterization of the protein phosphoamidates formed by a membrane bound adenylyl transferase reaction in Dictyostelium discoideum.
1. In this paper we report the results of studies on an adenylyl transferase (AyTase) activity from Dictyostelium discoideum. 2. Previous studies suggested that this activity catalyzed the transfer of AMP from ATP to a membrane protein to form a phosphoamidate reaction product. 3. In the present study we have isolated and characterized the product of the AyTase reaction and surprisingly found two AMP labeled products by SDS-gel-filtration HPLC. 4. The apparent molecular weights of these phosphoamidates were 13.5 and 1.5 kDa. 5. In addition, because the reaction product was rapidly degraded by a phosphoamidase, experiments were undertaken using AVAMP, a synthetic phosphoamidate, as an alternative phosphoamidase substrate, to trap the reaction products. 6. As the phosphoamidase activity could be inhibited by cAMP, cyclic formycin monophosphate, an analog of cAMP resistant to hydrolysis by cAMP phosphodiesterase, was also used to trap the products. 7. Both attempts at trapping failed. 8. A model for the AyTase reaction was developed to account for the failure to trap the products and the formation of two phosphoamidates.
Hadjimichael
J
J
Department of BioStructure and Function, University of Connecticut Health Center, Farmington 06030.
Rossomando
E F
EF
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001360:Avidin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004023:Dictyostelium; D004151:Dipeptides / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000378:metabolism*
D004151:Dipeptides; C069732:avidin-adenosine monophosphate conjugate; D001360:Avidin; C017330:N-formylmethionylphenylalanine; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; D006867:Hydrolases; C049021:phosphoamidase