Characterization of soil phosphorus by anion exchange resin adsorption and P32-equilibration

Summary Adsorption of phosphate by the anion-exchange resin Dowex-2 was investigated. The resin adsorbed small quantities of P from solution quantitatively. The rate of P-adsorption by resin agitated in solution was proportional to the P-concentration in solution, and was independent of the rate of diffusion of adsorbed P in the resin. When 1 g of soil was shaken continuously with 1 g of resin in 100 ml of water, the rate of P-adsorption by the resin was controlled by the rate of P-release from the soil. Quantities of P adsorbed from soil by resin after different lengths of time were less than those equilibrated with P³² during the same time intervals. The curves showing quantity of P adsorbed vs. time could be satisfactorily described by the hypothesis that there were three simultaneous reactions differing in rate, each reaction being first-order with respect to P. The same was true of the P³²-equilibration data, except that the rate of the slowest reaction was apparently independent of time. In a group of 16 soils, the correlation between P adsorbed by the resin in 2 hours and P-availability to plants in the greenhouse, measured by the isotope-dilution method of Fried and Dean, was 0.95. The corresponding correlation between P extracted by the 0.25N HCl — 0.03N NH₄F extractant of Bray and Kurtz was 0.91.Joint contribution from the Iowa Agricultural Experiment Station and the Eastern Soil and Water Management Section, U.S. Department of Agriculture. Journal Paper No. J-2639 of the Iowa Agricultural Experiment Station, Ames, Iowa. Project No. 1183.Graduate Assistants, Professor of Soils, and Associate Professor of Chemistry, respectively.     

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.     

P32 UPTAKE BY NUCLEI

1. A method for isolating nuclei in quantity from mammalian tissues is described. 2. The rate of uptake of radioactive phosphorus by nuclei is found to be quite rapid. The phosphorus was shown not to be taken up by exchange. 3. Nuclei of tumors accumulate more radioactive phosphorus than normal liver nuclei. This was shown to be due to mitotic activity and not a form of metabolism peculiar to tumor cells. 4. The specific activities of nuclei and cytoplasm are compared. 5. 60 to 70 per cent of the nuclear radioactive phosphorus is present as nucleoprotein from 1 hour to 5 days after it is administered. In the lymphoma nuclei 90–95 per cent of the phosphorus is in the nucleoprotein fraction from 1–5 days after it is administered. 6. The specific activities of the nucleoprotein, lipid, and acid-soluble fractions of liver and tumor nuclei are compared. 7. From the rate of P₃₂ uptake by nuclei it is calculated that a new lymphoma nucleus is synthesized on the average once every 27 hours. This is in agreement with the observed rate of growth of the tumor. 8. In the lymphoma nucleus it is calculated that 7 x 10⁴ molecules of tetranucleotide are synthesized per second. 9. Irradiation with 200 r. x-rays alters the distribution of P₃₂ in the lymphoma cell, markedly increasing the concentration in the nucleus shortly after irradiation. The P₃₂ concentration in the cytoplasm decreases with time after irradiation. It is suggested that the altered distribution is correlated with the inhibition of mitosis produced by the x-rays. 10. Continual synthesis of nucleoprotein takes place even in nuclei of cells which do not undergo mitosis.     

Assay of old-world screw-worm fly, Chrysomya bezziana, labelled with 32P

Techniques for ³²P labelling of larvae and adults of the screw-worm fly, Chrysomya bezziana, are described. Egg masses of labelled flies were readily identified. At the doses used for field releases, oviposition activity, fecundity, fertility and longevity of female flies were not adversely affected. Radioactive egg masses were recovered from sentinel animals following field release of labelled flies.

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Résumé Cet article décrit les techniques utilisées pour marquer avec le ³²p les larves et adultes de Chrysomya bezziana. Les pontes des mouches marquées ont été aisément identifiées. Aux doses utilisées pour le relâchement des insectes dans la nature, ni l'activité de ponte, ni la fécondité, fertilité et longévité des femelles n'ont été défavorablement modifiées. Les pontes radioactives ont été recueillies sur des animaux-pièges mis en place après le lâcher des mouches marquées.

     

32P-labeling of bovine tryptophanyl-tRNA synthetase with [32P]pyrophosphate

The covalent derivative of the tryptophanyl-tRNA synthetase obtained under the action of³²PP_i contains one mole of the covalently bound pyrophosphate (or 2 moles of orthophosphate) per mole of dimeric enzyme. Dephosphorylation with alkaline phosphatase causes practically no changes of enzymatic activity although the enzyme looses its ability to bind PP_i.Enzymes tryptophanyl-tRNA synthetase (EC 6.1.1.2), alkaline phosphatase (EC 3.1.3.1), inorganic pyrophosphatase (EC 3.6.1.1)     

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.