The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP.

A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic (32)P]AMP, and cyclic (32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of gamma-(32)P]ATP, gamma-(32)P]GTP, gamma-(32)P]ITP, and gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form 5'-(32)P]nucleoside monophosphates. The 5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic (32)P]AMP and cyclic (32)P]GMP are also prepared enzymatically from alpha-(32)P]ATP or alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the alpha-(32)P]nucleoside triphosphates. Methods for the use of the alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.