Isotopic labelling radioactive

Another widely used approach to the elucidation of metabolic sequences is to feed cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard radioactive isotopes, such as and stable heavy isotopes, such as or (Table 18.3). Because the chemical behavior of isotopically labeled compounds is rarely distinguishable from that of their unlabeled counterparts, isotopes provide reliable tags for observing metabolic changes. The metabolic fate of a radioactively labeled substance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 18.13). 

The application of substrates isotopically labeled in specific positions makes it possible to follow the fate of individual atoms during the microbial degradation of xenobiotics. Under optimal conditions, both the kinetics of the degradation, and the formation of metabolites may be followed— ideally when samples of the labeled metabolites are available. Many of the classical studies on the microbial metabolism of carbohydrates, carboxylic acids, and amino acids used radioactive... 

The analytical detectability applying a CL method should, in principle, be comparable to that obtained using radioactive labels, without all the disadvantages related to the use of isotopic labeling. In fact, assuming reasonable values for the quantum efficiency of the chemiluminescent reaction (Cl 0.01), for the overall photon collection efficiency of the optical system-CCD camera assembly (T) 0.01%), and for the intensity of the lowest detectable CL signal (about... 

Until recently, cell-free protein expression (also sometimes erroneously named in vitro protein expression) did not exhibit the productivity required for preparation of NMR samples, especially considering the high cost of using isotopically labeled starting material. Rather, it was exclusively used as an analytical tool that served to verify correct cloning or to study promotor sites. Because of the very low yields, detection of the expressed product usually required incorporation of a radioactive label (usually via 35S-methionine). 

Choosing a method to determine isotope effects on rate constants, and selecting a particular set of techniques and instrumentation, will very much depend on the rate and kind of reaction to be studied, (i.e. does the reaction occur in the gas, liquid, or solid phase , is it 1st or 2nd order , fast or slow , very fast or very slow , etc.), as well as on the kind and position of the isotopic label, the level of enrichment (which may vary from trace amounts, through natural abundance, to full isotopic substitution). Also, does the isotopic substitution employ stable isotopes or radioactive ones, etc. With such a variety of possibilities it is useless to attempt to generate methods that apply to all reactions. Instead we will resort to discussing a few examples of commonly encountered strategies used to study kinetic isotope effects. 

Commonly, in vitro determination of HDAC activity is a manual assay utilizing a coupled two-step process, including enzymatic deacetylation of a substrate followed by reaction termination and readout [10]. Assays utilize nuclear extracts and substrates containing labeled (radioactive or fluorescent) acetylated histones. For the isotope-based assays, the enzymes are incubated with acetate-radiolabled histones prepared from chicken reticulocytes or chemically [ Hjacetylated peptide substrates, and the enzymatic activity is determined by liquid scintillation counting [11]. Alternatively, histones may be obtained from cells following treatment with [ H]acetyl-CoA [12]. The caveats of these approaches include the variability of prelabeled acetylated core histones within preparations, potential high costs, their labor-intensive nature and the presence of radioactive waste. 

I.6.I. Radioactive Isotopes. The administration of isotopically labeled drugs is now commonplace in the experimental pharmacology of every new therapeutic agent (K18, M4) but has little or no clinical application. It often provides much of the initial information about the pharmacokinetics upon which subsequent clinical practice depends, but... 

The diffusion of alkyl ammonium ions into clay pellets has been studied by bringing the pellet into superficial contact with an isotopically labeled salt and then, after a suitable time, using a microtome to slice the pellet. The radioactivity is then measured in successive thin slices of the pellet. Assuming that Equation (62) describes the diffusion process, estimate how long it would take for 1% of the initial activity of each of the ions to appear in the 15th slice inward from the exposed surface of the dry clay if each slice is 40-pm thick. The diffusion coefficients for the methyl and trimethyl ammonium cations under these conditions are 7.03 x 10-12 and 2.65 x 10 " cm2 s, respectively.f... 

All radioactive materials should be stored in well-labeled, glass containers. The label must include your name, the type of isotope, the radioactive compound, the total amount of radioactivity, the specific activity, and the date of measurement. 

Of great importance, for these studies, was knowledge of both pool size and turnover. This knowledge permitted calculation of the specific activity of the applied isotopically labeled compound at any desired time. Thus, radioactivity, from a labeled compound applied to the endosperm and appearing in the shoot could be translated in amounts of compound moved from seed to shoot. 

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