Abstract
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.
Original language | English (US) |
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Pages (from-to) | 11-31 |
Number of pages | 21 |
Journal | BBA Section Nucleic Acids And Protein Synthesis |
Volume | 562 |
Issue number | 1 |
DOIs | |
State | Published - Mar 28 1979 |
Bibliographical note
Funding Information:The authors are deeply indebted to Dr. Giinter Schultz of the Pharma-kologisches Institut der Universit~it Heidelberg, Heidelberg, Germany, for many helpful suggestions in the course of the development of this procedure. The authors wish to thank Dr. David L. Garbers, of the Department of Pharmacology, Vanderbilt University, for the generous gift of partially purified guanylate cyclase and Dr. Dixie Frederickson, Department of Biochemistry, Vanderbilt University, for the generous gift of myosin. We are also indebted to Drs. Jack N. Wells and George Kramer of the Pharmacology Department, Vanderbilt University for conducting a number of phosphodiesterase and cyclic AMP binding assays to check the quality of the cyclic \[32p\]AMPa nd cyclic \[32p\]GMP produced. We wish to acknowledge the excellent technical assistance of Ms. Janette Welden and Mr. Tony Ross. The work was supported by grants from the United States Public Health Service, AM 18185, AM 21170, AM 07462, and HL 19325.
Keywords
- (Radiolabeled)
- Cyclic nucleotide
- Nucleotide derivative