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.
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000263:Adenylate Kinase / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis*; D006152:Cyclic GMP / Q000096:biosynthesis*; D003851:Deoxyribonucleases / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000096:biosynthesis*; D006162:Guanylate Cyclase / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D010761:Phosphorus Radioisotopes; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012265:Ribonucleotides / Q000096:biosynthesis*
D003854:Deoxyribonucleotides; D010761:Phosphorus Radioisotopes; D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011770:Pyruvate Kinase; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D000263:Adenylate Kinase; D003851:Deoxyribonucleases; D012260:Ribonucleases; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D006152:Cyclic GMP