Radioactivity assay, activation

The post-bombardment processing of the activated sample may follow either a nondestructive assay of the radioactivity in the sample (gamma-ray scintillation spectrometry is used most often for this) or a chemical processing of the sample prior to the radioactivity assay. Techniques involving either precipitation, electrodeposition. solvent extraction, and ion exchange or some combination of these form the basts of the radio-chemical separation techniques used in activation analysis. 

The most sensitive assay method for PE activity is to use, as substjgte, pectin esterified biologically (31) or chemically (32) with C methanol. In studies by Gessner (29) and Bartolome and Hoff (30) the substrate and enzyme were precipitated from the reacj on mixture with acidified ethanol or TCA before activity of the C methanol in the supernate was determined. The radioactive assay method was about 100 times as sensitive as the titrimetric method for PE activity (32). 

An assessment of the methods for determining active centre concentrations has been made by Schnecko and Kern [213], and they conclude that radioactive assay is more reliable than indirect kinetic methods. Propene and butene-1 polymerized by TiClj/AlEtjCl on termination with iodine or BuO H gave comparable extrapolated values of ca. 0.5% for catalyst efficiency. This is lower than earlier estimates but higher than the very low values (0.1%) obtained by Coover and Guillet [121], although the shapes of the curves obtained by the two groups were very similar. 

The sialyltransferase assay is usually carried out with radioactive CMP-Neu5Ac and the sialic acid acceptor, followed by identification of the radioactive product. As this product is often membrane-bound and the radioactive cosubstrate is expensive, a non-radioactive assay was developed [625], in which CMP released from CMP-Neu5Ac by the transfer of Neu5Ac is determined with HPLC (see section 5.3.2). Since CMP-Neu5Ac can be analyzed in the same run, this test is also suited for the activity of the CMP-Neu5Ac synthase described above. 

A great number of methods are availaible for assaying activities of lipolytic enzymes(l). The most commonly used techniques include titrimetric, radioactive, or optical methods. Most of these techniques are time-consuming and/or suffer from several shortcomings, such as poor reproducibility, lack of sensitivity, or the production of radioactive waste. Here, we describe the synthesis and application of a new class of fluorogenic substrates that are useful substrates for the fast and accurate determination of activity and stereoselectivity of lipolytic enzymes in aqueous media and organic solvents (4, 5)... 

Aliquots of incubation medium were taken each time and centrifuged at 500 xg for 2 min. The supernatants were recovered and saved. The cells were resuspended in Hanks buffer and centrifuged at 3,000 xg for 5 min. Lipids from the cellular pellets were extracted by the method of Folch-Pi et al. (1957), saponified, ester-ified, and assayed for A9 desaturase activity with TLC-AgN03 (De Tomas and Brenner, 1964). Radioactivity assays was determined in a Packard scintillation spectrophotometer. 

The number of active carbon atoms in each degradation product was calculated from the radioactive assay data, on the assumption that radioactive Qg-hydroxy acid contains 6 labeled carbon atoms when derived from acetate- C, and a single labeled carbon atom from methyl-labeled L-methionine as illustrated below... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Isotope Dilution Assay. An isotope dilution assay for biotin, based on the high affinity of avidin for the ureido group of biotin, compares the binding of radioactive biotin and nonradio active biotin with avidin. This method is sensitive to a level of 1—10 ng biotin (82—84), and the radiotracers typically used are p C]biotin (83), [3H]biotin (84,85) or an I-labeled biotin derivative (86). A variation of this approach uses I-labeled avidin (87) for the assay. 

Radioisotope dilution assays are based on the principle of competition between radioactive labeled ( Co) vitamin B 2 and cobalamins extracted from matrices for binding sites on the intrinsic factor (a glycoprotein). Binding is in proportion to the concentration of the radioactive and nonradio active B 2 with the concentration of intrinsic factor as the limiting factor. Free cobalamins are separated from those bound on the intrinsic factor by absorption... 

The activities of S6K1 or S6K2 may be assessed by direct (radioactive) kinase assay using a 32-amino acid peptide (Moule et al, 1995), 40 S ribosomal subunits (Chen and Blenis, 1990), or S6, expressed as a fusion protein with GST (Patti et al, 1998). 

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