6-labeled 1-phosphate

Figure 1.72 Cystamine may be used to label phosphate groups, such as at the 5 -end of oligonucleotides, via a carbodiimide reaction using EDC. The resultant phosphoramidate linkage is a common way to modify oligonucleotides at the 5 -end.

Treatment with hot organic solvents was the next step in the tissue fractionation, to remove lipid-phosphorous and breakdown lipid-protein interactions. In the Schneider procedure, nucleic acids were then extracted in hot dilute trichloroacetic or perchloric acid, leaving a protein residue with any phosphoprotein links still intact. This method was to become particularly useful when 3H thymidine became the preferred label for DNA in the early 1960s. For investigations where both RNA and DNA were to be examined the Schmidt-Thannhauser process was often chosen. Here the lipid-extracted material was hydrolyzed with dilute sodium hydroxide releasing RNA nucleotides and any hydroxyamino acid bound phosphorus. DNA could be precipitated from the extract but the presence in the alkaline hydrolysate of the highly labeled phosphate released from phosphoprotein complicated... 

In order to measure, by the methods of isotopic dilution the mass concentration of the orthophosphate ion PO4-, in an aqueous solution. 3 mg of labelled phosphate ion 32PC>4 (32P being a /3-emitter whose half-life is 14 days), is added to 1 g of the sample solution. 

The alkaline phosphatase of E. coli is a dimer of 449-residue subunits which requires Zn2+, is allo-sterically activated by Mg2+, and has a pH optimum above 8.667/708 711 At a pH of 4, incubation of the enzyme with inorganic phosphate leads to formation of a phosphoenzyme. Using 32P-labeled phosphate, it was established that the phosphate becomes attached in ester linkages to serine 102. The same active site sequence Asp-Ser-Ala is found in mammalian alkaline phosphatases. These results, as well as the stereochemical arguments given in Section 2, suggest a double-displacement mechanism of Eq. 12-38 ... 

Triply labeled phosphate has been employed to elucidate the steric course of a number of phosphoryl transfer reactions the topic has been reviewed 165>166>, and we shall here present only one example, concerned with the stereochemistry of cycliza-tion of ADP to cAMP 167,168) and the reverse reaction 169,170). 

The incorporation of labeled phosphate in PI to generate PIP2 in normal as well as in 7315a PRL-secreting tumor cells appears to be inhibited by DA [109,110]. Whether this effect is related to the hydrolysis of PIP2 into IP3 and consequent Ca2+ mobilization from endoplasmic reticulum remains to be shown. 

There is general agreement that LHRH stimulates the incorporation of labeled phosphate into the phosphoinositides PI, PIP and PIP2 [111-115]. This has also been... 

When a radioactivity-labeled phosphate was transferred by the ser/threo kinase to the filter-bound histone, light emitted was measured in a scintillation counter. 

Yakhnina (Yl) reported that the rate of phosphoprotein circulation in gastric mucosa of cats injected with labeled phosphates was elevated upon histamine stimulation of gastric secretion. She considered it probable that phosphoprotein actively participates in gastric secretion. 

More direct evidence that cellular actin is ADP-ribosylated in intact cells can be obtained, when cells are metabolically labeled with [ P]orthophosphoric acid or with [2- H]adenine. Radioactively labeled phosphate/adenine is incorporated into cellular NAD which is the cosubstrate for C2l-catalyzed ADP-ribosylation. 

Recent isotope incorporation studies by a number of workers have indicated increased turnover of muscle membrane lipids following denervation. These include increased incorporation of labeled phosphate and glycerol into glycerophosphatides (B17), increased turnover of... 

Duplex surface coverages (as determined by labeling, phosphate... 

In the Maxam and Gilbert method (M2) the starting point is a defined DNA restriction fragment. The DNA strand to be sequenced must be radioactively labeled at one end with a 32P-labeled phosphate group. This DNA can be single stranded or of duplex form. The base-specific cleavages depend upon the following points ... 

D6. De Verdier, C. H., The incorporation of labelled phosphate into different compounds in the rat kidney the effe of parafh)nxiid extract on an in vivo system. Acta Physiol. Scand. 39, 1-11 (1967). 

The rate of phosphate binding to EPSP synthase was measured by a single-turnover experiment in the reverse reaction shown in Fig. 10. The experiment was initiated by mixing an excess of enzyme and EPSP with a trace of labeled phosphate (<1 fiM). Under these conditions, the rate of formation of PEP was limited by the rate of phosphate binding to the enzyme-EPSP complex. However, in the absence of unlabeled PEP, the reaction did not go to completion. Successful execution of this experiment required the addition of the unlabeled reaction product (PEP) in order to ensure that the release of radiolabeled PEP was irreversible. In the absence of unlabeled PEP, the reaction came to equilibrium short of complete conversion of radiolabeled phosphate to PEP. The addition of unlabeled PEP pulled the reaction to completion by dilution of the radiolabeled PEP. Computer simulation was required to analyze quantitatively the reaction time course. Conventional data fitting to the time dependence of the reaction gives a rate of approximately 0.035 sec . The simple interpretation would then lead to calculation of a second-order rate constant for phosphate... 

Hladik WB, Ponto JA, Lentle BC, Laven DL (1987) Iatrogenic alterations in the biodistribution of radiotracers as a result of drug therapy reported instances. In Hladik WB III, Saha GB, Study KT (eds) Essentials of nuclear medicine science. Williams Wilkins, Baltimore, pp 193-202 International Commission on Radiological Protection (1987 a) Technetium-labelled phosphates and phosphonates. In Annals of the ICRP, radiation dose to patients from radiopharmaceuticals, biokinetic models and data. ICRP publication 53, vol 18, no 1-4. Pergamon, Oxford, pp 213-215... 

Conklin JJ, Alderson PO, Zizic TM et al (1983) Comparison of bone scan and radiograph sensitivity in the detection of steroid-induced ischemic necrosis of bones. Radiology 147 221-226 Council of Europe (2005) Technetium " Tc medronate injection. In European Pharmacopeia 5.0, monograph no 641. Council of Europe, Maisonneuve, Sainte-Ruffine, p 1219 Davis MA, Jones AG (1976) Comparison of " Tc-labeled phosphate and phosphonate agents for skeletal imaging. Semin Nucl Med 6 19-31... 

FIGURE 2 Phosphoamino add analysis of individual phospho-peptides (A-F) from p-labeled LC20. Pj, Ser-P, and Thr-P are labeled inorganic phosphate, serine-phosphate, and threonine-phosphate, respectively, and Pep-P is the unhydrolyzed P-labeled phosphate. From Erdddi et al. (1988a, Rg. 5, p. 587). 

On oxidation of the sulphide PhSCH3191a in [lsO]-labelled phosphate buffer solution, only 1.3% of the H2lsO used has been incorporated into the produced sulphoxide... 

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