Radioisotope labeling experiments

The most frequently used combination of radioisotopes in double labeling experiments is tritium and 14C. Ideally the ratio of the activities in the corresponding channels (Fig. 7.16) should to be high enough to insure the carbon counts in the tritium channel are negligible compared to tritium counts. Usually this is not achieved and a correction must be applied. 

This was demonstrated for Co(C204)3 and Cr(C204)3 in one of the earliest radioisotopic exchange experiments. Exchange of these ions with "C-labeled C204 is very much slower than racemization F. A. Long, J. Amer. Chem. Soc. 61, 571 (1939) 63, 1353 (1941). 

Similar experiments with dual radioisotopic labeling show that there is a small manganese(II) ion excess in the manganese(II) ion/calcium-montmorillonite (labeled with 54Mn and 45Ca isotopes) interfacial reaction. It means that there is an adsorption reaction besides ion exchange, but it has a very low contribution. For this reason, the presence of adsorption can be observed, but its quantitative treatment is difficult. 

Counting more than One Isotope in a Sample The basic liquid scintillation counter with coincidence circuitry can only be used to count samples containing one type of isotope. Many experiments in Biochemistry require the counting of just one isotope however, more valuable experiments can be performed if two radioisotopes can be simultaneously counted in a single sample (double-labeling experiments). The basic scintillation counter previously described has no means of discriminating between electrical pulses of different energies. 

Several plants of the genus Aphelandra (Acanthaceae) also produce spermine alkaloids, and among them, aphelandrine is the main alkaloid. When radioisotope-labeled potential precursors of aphelandrine were fed to Aphelandra tetragona, it was shown that putrescine, spermidine, and cinnamic acid were incorporated into aphelandrine. It was not clear whether spermine was incorporated directly into aphelandrine. Spermine might have been metabolized to putrescine and spermidine before incorporation. It was clarified in this experiment that methionine was the precursor of the 3-aminopropyl moiety of spermidine and spermine [9]. 

This approach has been successful in a number of affinity labeling experiments. The basic protocol requires using both a pep-tRNA and an aa-tRNA. One of them has a radioactive amino acid (alternatively, the two amino acid moieties may be labeled with different radioisotopes) the other has an electrophilic or photoactivatable group attached to the side chain or the -amino nitrogen of the amino acid. The pep-tRNA is bound to ribosomes under conditions that favor binding to the P site. If this tRNA contains the affinity or photoaffinity label, reaction with the ribosomal components is allowed to proceed. Then the aa-tRNA is added to the incubation, and a peptide bond is allowed to form between the two aminoacyl moieties. Alternatively, the reactive probe may be attached to the aa-tRNA. Since peptide bond formation follows rapidly upon binding, the covalent reaction of the probe with ribosomal components presumably occurs after peptide transfer. 

Molybdenum consists of seven stable isotopes, Mo, Mo, Mo, Mo, Mo, Mo, and °°Mo, with average natural abundances of 14.77, 9.23, 15.90, 16.68, 9.56, 24.19, and 9.67%, respectively. Half-lives of molybdenum radioisotopes are in general not long enough (<67 h) for kinetic studies or intrinsic labeling experiments. So far, only a single study has been conducted to compare molybdenum absorption from extrinsically and intrinsically labeled foods [282]. Absorption of both labels was statistically not different for kale. For soy, however, the intrinsic label was less well absorbed and less well excreted than the extrinsic label given as Mo-molybdate. 

For radioautography to be a useful tool in correlation of structure and function, the compound visualized must be well defined in chemical terms. This is best achieved when the precursor used will give a single product, or when the labeled compound used is not further metabolized. The identification of the product can be verified by radiochemical and chromatographic analysis of a portion of tissue adjacent to that used for radioautography. In addition, and especially in long-term experiments, it is necessary to establish the stability of the radioisotope label in the compound to be localized. 

Evidence obtained with radioisotopically labeled tryptophan as well as with certain tryptophan analogues have established that tryptophan is a precursor of the actinomycin chromophore (Katz, 196O Sivak, Meloni, Nobili and Katz, 1962 Sivak and Katz, I962). In experiments with benzene ring-labeled tryptophan it was shown that the C-label of the amino acid was efficiently (11.7%) and preferentially incorporated into the phenoxazinone moiety of actinomycin (Table 3). The chromophore derivatives, actinocinin and desaminoactinocylthreonine dimethyl ester, obtained from actinomycin by acid hydrolysis (Brockmann and Grone,... 

In the experiments with acetic acid labelled radioisotopically and fed to ani-mals, it has been established that the cholesterol carbon framework is made up entirely of the acetic acid carbon. 

Virtually all biosynthetic studies with molluscs have been conducted using precursors labelled with radioisotopes. In general, experiments are carried out... 

Measurement of the serum concentrations of administered antibodies is a general tool to evaluate their persistence in circulation. This is usually performed by introducing a sufficient amount of the test antibody either by the intravenous or by the intraperi-toneal routes (see Note 3), in a quantity that can be easily detected and quantified in serum samples, even after a two log reduction in concentration. The antibody tracer can be labeled with radioisotope which permits direct quantification in serum samples. To minimize radioactive isotope use, we use an antibody tracer that is unmodified or labeled with biotin or other derivation chemistries, and then determine its serum concentrations by ELISA techniques. We commonly inject 100 xg of the test antibody in a 200 xl volume of phosphate-buffered saline into each mouse intraperitoneally (i.p.) (see Note 4). This amount can vary depending on the goals of the experiment and the sensitivity of the detection method. A minimum of five inbred mice, sex-matched and age-matched, at 8-16 weeks of age are recommended for each antibody to be tested. 

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