A simple enzymic method for the synthesis of adenosine 5''-[alpha-32P]triphosphate on a preparative scale.

A simple, rapid and inexpensive method is described for the enzymic synthesis of [alpha-32P]ATP from [32P]Pi on a preparative scale with an overall yield of 53%. The final product contained all of the detectable radioactivity (less than 99.9%) in the alpha position and has been shown to behave identically with commerically availabe [alpha-32P]ATP during the synthesis of 3':5'-cyclic AMP in the reaction catalysed by adenylate cyclase.     

Isolation of 1-[32P]phosphohistidine from rat-liver cell sap after incubation with [32P]adenosine triphosphate

High molecular weight material, obtained from rat-liver cell sap by chromatography on Sephadex G-50, was incubated for 15 sec at pH 7.4 and 0° with highly labelled [³²P]ATP. Incubation was terminated by addition of NaOH to a final concentration of 0.1 M. Afer removal of excess [³²P]ATP on Sephadex G-50 the ³²P-labelled, high molecular weight material was precipitated with acetone and hydrolyzed in 3 M KOH at 100° for 3 h.In addition to the 3-[³²P]phosphohistidine previously isolated, ³²P-labelled material was obtained which co-migrated with synthetic 1-phosphohistidine when the alkaline hydrolysate was chromatographed on Dowex 1. The identity of this material with 1-phosphohistidine was established by paper electrophoresis at five different pH levels, paper chromatography in two systems, rechromatography on Dowex 1 and a study of its rate of hydrolysis in acid molybdate.     

Carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate

We found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed us to synthesize in a single-step procedure carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[gamma-32P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue.     

Synthesis of [gamma-32P]thiamine triphosphate

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%.     

A simple enzymic method for the synthesis of [32P]phosphoenolpyruvate

A rapid and simple enzymic method is described for the synthesis of [³²P]phosphoenolpyruvate from [³²P]P_i, with a reproducible yield of 74%. The final product was shown to be a good substrate for pyruvate kinase (EC 2.7.1.40).     

A simple method for the preparation of [beta-32P]purine nucleoside triphosphase.

A rapid, simple and inexpensive procedure is described for the preparation of purine ribo-and deoxyribonucleoside triphosphates specifically and highly radiolabeled with [32P]phosphate in the beta position. The method involves two successive enzymatic reactions: conversion of donor [gamma-32P]ATP in the presence of an excess of acceptor 5'-mononucleotide to the 5'-diphosphates by myokinase or guanosine 5'-monophosphate kinase followed by phosphorylation with pyruvate kinase to yield 5'-triphosphates.     

The rapid, simple and improved preparation of high specific activity alpha-[32P]dATP and alpha-[32P]ATP.

An improved method is described for the rapid and simple preparation of alpha-[32P]dATP and alpha-[32P]ATP from 32Pi in good yields and with specific activities from 20 - 150 Ci/mmol. The two-step procedure involves the chemical synthesis of the mononucleotide followed by its enzymic conversion to the triphosphate with myokinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) in the presence of trace amounts of dATP or ATP to prime the reaction. The two steps are carried out in the same reaction flask and the only purification step required is a step-wise elution from a column of DEAE-cellulose.     

A calcium ion-dependent adenosine triphosphate pyrophosphohydrolase in plasma membrane from rat liver. Demonstration that the adenosine triphosphate analogues adenosine 5''-[betagamma-imido]triphosphate and adenosine 5''-[betagamma-methylene]-triphosphate are substrates for the enzyme.

An ATP pyrophosphohydrolase in a rat liver plasma-membrane subfraction was studied with respect to specific Ca2+ activation of the beta-phosphate bond hydrolysis. ATP and, in addition, adenosine 5'-[betagamma-imido]triphosphate and adenosine 5'-[betagamma-methlylene]triphosphate were substrates for Ca2+-stimulated enzymic hydrolysis of the beta-phosphate bond. A 15-fold activation was observed by raising the free Ca2+ concentration from 10(-7) to 10(-5) M. Mg2+ had little effect. Solubilization in 1% deoxycholate and partial purification on a sucrose density gradient resulted in a 5-fold increase in specific activity with unaltered Ca2+-stimulation pattern. The possible importance of the enzyme in Ca2+ transport is discussed.     

An enzymatic method for [32P]phosphoenolpyruvate synthesis

A convenient method for the enzymatic synthesis of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-³²P]ATP to [³²P]phosphoenolpyruvate, which was subsequently separated from residual [γ-³²P]ATP and $\text{[}{}^{32}\text{P}\text{]P}{}_{\mathrm{<mtext>i</mtext>}}$ by chromatography on AG-1-X8-bicarbonate resin.     

A procedure for the preparation of [32P]phosphatidic acid

The phosphorylation procedure of F. Cramer, W. Rittersdorf, and W. Bohm [(1961) Chem. Ber. 654, 180] using bis(triethylammonium) phosphate and trichloroacetonitrile was shown to be effective in the synthesis of [32P]phosphatidic acid. From diacylglyceride and 0.5 mCi H(3)32PO4, 25-50 microCi of labeled material (sp act = 1 mCi/mumol) can be prepared in 2 h. The product was shown to be radiochemically pure by both TLC and HPLC. L- and DL-[32P]dipalmitoyl phosphatidic acid prepared using this procedure were shown to be hydrolyzed by rat liver microsomes at approximately the same rates.     

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.     

Preparation of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine by using soya beans.

This method describes a procedure that can be carried out easily to obtain large amounts of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine. The method involves germinating soya beans in the presence of [32P]Pi. The yield was 0.58% for [P]phosphatidylcholine and 0.52% for [32P]lysophosphatidylcholine, and the specific radioactivity of both was 10(7) d.p.m./mumol.     

Enzymatic synthesis of [beta-32P]ADP using adenylate kinase and [gamma-32P]ATP

An enzymatic method for the synthesis of [beta-32P]ADP from [gamma-32P]ATP is described. This substrate is required for the assay of ADPase and is not commercially available. The method described results in a preparation of [beta-32P]ADP of high purity with a yield of approximately 40% the theoretical obtainable.