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.     

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.     

5-Methyltryptamine decreases net accumulation of 32P into the polyphosphoinositides from [gamma-32P]ATP in a cell-free system from blowfly salivary glands. Activation of breakdown of the newly synthesized [32P]polyphosphoinositides

Incubation of blowfly salivary gland homogenates with 30 microM [gamma-32P]ATP resulted in a rapid, Mg2+-dependent, synthesis of [32P]polyphosphoinositides and [32P]phosphatidic acid. 5-Methyltryptamine, in the presence of 10 microM guanosine 5'-(3-O-thio)trisphosphate, reduced the net accumulation of 32P label into phosphatidylinositol-4,5-P2 and phosphatidylinositol-4-P by 35 and 20%, respectively. 5-Methyltryptamine did not affect synthesis of [32P]phosphatidic acid. Phosphorylation of polyphosphoinositides was not affected by 5-methyltryptamine. In membranes labeled in vitro with [gamma-32P]ATP, 5-methyltryptamine stimulated a rapid breakdown of the [32P]polyphosphoinositides. These results indicate that in blowfly salivary gland homogenates, hormone stimulates breakdown of the newly synthesized polyphosphoinositides. In the presence of hormone, the rate of polyphosphoinositide synthesis does not compensate for the rate of polyphosphoinositide degradation.     

Mitochondria with impaired phosphate transport: 32P uptake studies.

Rat liver mitochondria which have been exposed to 0.15 M NaC1 at 35 degrees C for 15 min subsequently take up 32Pi from an external medium only to about 5% of the extent of uptake by control mitochondria. The volume into which 32Pi distributes in a pellet of such "aged" mitochondia is less than that available to 3H2O but is greater than that available to [3H]sucrose. Mitochondria treated in this manner cannot therefore accumulate Pi although limited penetration of the inner membrane can occur. These results confirm earlier findings by indirect methods (Williams, G. R. & Orr, J. L.: Dynamics of energy-transducing membranes (Ernster, L., Estabrook, R. W. & Slater, E. C., eds), Elsevier Scientific Publishing Company, Amsterdam, Netherlands, pp. 497-508 (1974).     

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)