Radioactive phosphate

Many lanthanide and actinide minerals in nature are phosphates. Examples are monazite (CeP04), LaP04, NdP04, and ThP04. In monazite, substitution of radioactive elements is possible, e.g., Ce can be replaced partially by Th, and that makes the material radioactive. Phosphates of other actinides, such as plutonium phosphate (PUPO4), fall into the same category. 

Phosphorylase phosphatase (EC 3.1.3.17 phosphorylase phosphohydrol-ase) catalyses the conversion of phosphorylase a to phosphorylase b with the release of inorganic phosphate The reaction can be followed by the disappearance of phosphorylase a activity or by the release of radioactive phosphate from P-labelled phosphorylase a [167]. 

Q.Convert the insert io the DNA probe At this point, the test tube contains a large quanlitj of the insert. The goal is to convert the insert to a form usable as a probe. T4 polynucleotide kinase is used to catalyze the attachment of radioactive phosphate to the ends of the DMA ... 

Note that any NAD" containing solutions must be protected from light, which is easily done by wrapping containers with aluminum foil. Radiolabelled P-NAD" is either synthesized from [a- P]ATP by the method of Cassel and Pfeuffer (1978) or purchased and stored frozen. Although the half-life of the radioactive phosphate is approximately two weeks, we do not use P-NAD" longer than three to four weeks after preparation because of radiolysis. 

Incubation of guinea pig ear or human epidermis with radioactive phosphate and isolation of the resulting phosphate-containing compounds by chromatography and radioautography disclosed about 20 labeled... 

Fig. 7.18. End-labeling can be achieved by different approaches. Kinasing (I) is most effective with 5 -OH overhangs. In the presence of excess ADP, radioactive phosphate can also be exchanged with a 5 phosphate. Whereas kinasing introduces at most one label per DNA end, tailing (II) allows multiple labels to be introduced (Co is necessary with recessed ends). Fill-in of restriction sites (III) also yields 3 -end labeling. RTase or sequenase should then be used. Oligomers are required if the 5 -end is recessed (IV).

In the Moore and Sharp19 procedure outlined below a radioactive phosphate is inserted at the junction by labelling the 3 -RNA using T4 polynucleotide kinase and [y-32P]ATP, but the ligation approach is useful for many other types of modifications. Other applications include ... 

Insertion of a radioactive phosphate at a specific site for label-transfer by UV crosslinking.23... 

Administration of glucagon, which lowers HMG-CoA reductase activity, to rats fed P-phosphate increases the amount of radioactive phosphate bound to HMG-CoA reductase compared to controls [193],... 

A scientist added phosphate labeled with radioactive phosphorus ( P) to a bacterial culture growing anaerobically (without O2). She then purified all the compounds produced during glycolysis. Look carefully at the steps of the pathway. Predict which of the intermediates of the pathway would be the first one to contain radioactive phosphate. On which carbon of this compound would you expect to find the radioactive phosphate ... 

With full radioactivity, phosphate glasses showed strong devitrification at 500 C with deterioration of leach resistance. Under the same conditions borosilicate glasses did not devitrify within 7 months [B3]. Recent investigations show that extensive additional tests under hydrothermal conditions are required to simulate underground storage conditions. 

Aq soln of mixed radioactive phosphates with a pH range of 3.0-6.0. Contains radioactive monobasic sodium phosphate (NaH,31P01) and radioactive dibasic sodium phosphate (NajH PO ). MP is a pure beta emitter with a half -life of 14.3 days. 

Isotope exchange is most easily understood in relation to a specific example. Consider the phosphorylation of fmctose-6-P by ATP, a reaction catalyzed by Lactobacillusplantarum phosphofmctokinase, which proceeds via an Ordered Bi Bi mechanism at neutral pH (Simon Hoffer, 1978). If the coenzyme ATP, labeled in the y-position with P, is used for the equilibrium exchange studies, the terminal radioactive phosphate group will be transferred from ATP to fiuctose-6-P ... 

These three methods are commonly used the detection of radioactive phosphate containing [24], the colorimetric detection of a complex of phosphomolybdate and malachite green [25], and the colorimetric detection of a phosphorylated product obtained by a secondary enzymatic phosphate transfer using purine ribonucleoside phosphorylase (PNP) [26] (Figure 2). 

Although it is outside the scope of this chapter to even begin to expose the wealth of information concerning the formation of polymeric DNA and RNA beyond that which has already been mentioned, it is, nonetheless, important to recognize that the overall reaction catalyzed by DNA polymerase enzymes is to take a growing deoxyribonucleotide monophosphate polymer and to add to it a deoxyribonucleo-tide triphosphate and to eliminate two linked phosphate units, that is, PiO or its equivalent (commonly written as PP). Further, it is important to note that in principle and in practice, the polymerization process is reversible. And, both in principle and in practice, it is common to find the phosphate units scrambled. Indeed, it is presumed that this is one of the ways of introducing radioactive phosphate into deoxyribonucleotide triphosphates. ... 

The identirtcation of phosphoryiated residue(s) requires a highly purified protein in sufficient amounts (1 nmol 50 ig) to reach the final microsequencing step of the labeled peptide(s) successfully. Consequently, labeling procedures have to be scaled up accordingly, and it is advisable at this stage to use radioactive phosphate as a tracer. 

Figure 28.6 The disappearance of radioactive phosphate from a solution containing phosphate ions in equilibrium with those in solid calcium hydroxyapatite. Step I takes place in minutes, step II in hours and step III in days...

We mentioned at the beginning of the chapter that transport occurs from the top downward and also in the reverse direction in the vascular system. A simple experiment provides us with the evidence for this statement (Fig. 213). A maize plant is placed in nutrient solutions in such a way that one half of its root system is immersed in nutrient solution A and the other half in nutrient solution B. Compounds containing radioactive phosphate, P re present in nutrient solution B. At the beginning of the... 

More recently, a number of reports have appeared demonstrating that phosphorylation could be measured in a microarray format using a known kinase-substrate pair. Two different detection approaches have generally been used, the first makes use of [y- / P]-ATP to label the immobilized substrate with a radioactive phosphate, which can be detected by autoradiography, phosphor imager, or silver staining [9,13,75-79]. The second detection method relies on phosphospecific antibodies, which are fluorescently labeled [13,51,58,76,80]. While the fluorescent detection may be preferable as it avoids working with radioactive ATP, it has been shown that only monoclonal anti-phosphotyrosine antibodies showed reliable results [76]. Alternatively, fluorescently labeled phosphor-chelators have been used to detect the phosphorylated peptide in an array [81]. Preliminary results have also been reported for mass spectrometry detection and surface plasmon detection (vide infra). 

Five minutes after intravenous injection of radioactive phosphate, Kalckar and his associates (102) found the labile phosphorus of adenyl pyrophosphate present in rabbit liver to have a specific activity of 83% of the corresponding value for inorganic intracellular phosphorus. In interpreting this and similar figures, we must take into account that in the course of five minutes the plasma activity declines considerably, very highly active phosphate penetrating the liver cells in an early phase of the... 

The specific activity of phosphatides extracted from rabbit and rat kidney is lower than that of phosphatides from small intestine and liver at early intervals after administration of radioactive phosphate. After the lapse of six hours the same result was obtained in experiments with dogs, but after eighteen hours the specific activity of the kidney phosphatide remained lower than that of liver and about equal to that of small intestine. At 98 hours the specific activities of the phosphatides in all three tissues wore roughly the same (32). Acidosis induced by ingestion of ammonium chloride increases turnover of kidney phosphatides, according to Weiss-berger (181). Administration of phlorizin did not influence turnover rate of phospatides in kidneys of the rat (182). 

The question whether a tissue can synthesize phosphatides independently or whether it acquires phosphatides from the plasma only after their formation by a more active tissue, was answered by Chaikoff et al. (62) in the following manner. A sciatic nerve of a dog stripped free of all adipose and connective tissue and weighing 300 mg. was placed in 5 ml. of Ringer solution containing radioactive phosphate. For control purposes the adipose-connective tissue surrounding the nerve was treated in a similar way. Conversion of radiophosphate from the Binger solution into radiophosphatide by the nerve was found to be considerable, as is seen in Table XVIII. These experiments show that the nerve process, separated from the nerve cell body, can form phosphatides from inorganic phosphate. 

Lindahl and associates (115a) found recently that radioactive phosphate introduced into the cells of bakers yeast was incorporated into the coenzyme molecule. The rate of this process was under certain conditions dependent on the rate of metabolism but the phosphate exchange also took place in the absence of exogenous substrate at low temperature (-1-4°C.), though at a very slow rate. 

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