ENZYMATIC PREPARATION OF [ALPHA-P-32] NUCLEOSIDE TRIPHOSPHATES, CYCLIC [P-32] AMP, AND CYCLIC [P-32] GMP

A method has been developed for the enzymatic preparation of α-32P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP of high specific radioactivity and in high yield from 32Pi. The method also enables the preparation of [γ-32P]ATP, [γ-32P]GTP, [γ-32P]ITP, and [γ-32P]-dATP of very high specific activity and in high yield. The preparation of the various [α-32P]nucleoside triphosphates relies on the phosphorylation of the respective 3′-nucleoside monophosphates with [γ-32P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5′-32P]nucleoside monophosphates. The [5′-32P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [α-32P]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 [32P]AMP and cyclic [32P]GMP are also prepared enzymatically from [α-32P]ATP or [α-32P]GTP by partially purified preparations of adenylate or guanylate cyclase. With the exception of the cyclases, all enzymes used are commercially available. The specific activity of 32P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [α-32P]ATP and from 5800 to 6500 Ci/mmol for [γ-32P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [α-32P]nucleoside triphosphates. Methods for the use of the [α-32P]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 32P-labeled compounds of biological interest. Moreover, the [α-32P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [γ-32P]nucleoside triphosphates should be useful in the study of phosphate transfer systems. © 1979.