Radioactive substrates

Fields et al. (1981) eliminated the separation step by using glutamate decarboxylase which converts (in a scintillation vial) L-f C]-glutamic acid to C02 which is released from the aqueous phase. Detectability is about 100-fold higher than with RIA. 

Bovine serum albumin (BSA)-antibody conjugates in the last step of the EIA, followed by anti-BSA antibodies conjugated with enzyme can also be used (Guesdon et al., 1983). 

Non-specific reactions in EIA can be due to a variety of mechanisms, many of which are discussed in detail elsewhere (Section 6.1.6.3, Section 8.6, Section 17.3.4). Non-specific reactions enhance background levels ( noise of experiment ) and have, therefore, a direct impact on the detectability. 

Schuit et al. (1981) investigated the specificity of 29 fluorochrome-conjugated antisera against human IgG and found half of these commercial preparations unsatisfactory, indicating the frequent absence of adequate quality control. The adequacy of the antiserum should, therefore, be established by strict testing. 

One or several of the following approaches may be taken to eliminate or reduce interference by RF (i) use of F(ab )2 (RF reacts only with the Fc of antibodies) (ii) removal of RF with an excess of Fc of the species used as antibodies (Winchester, 1980) (iii) addition of an excess of normal IgG or heat aggregated IgG (0.3 mg/ml) (Millan and Stigbrand, 1981) this method is very simple but can 

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N-(hexadecanoyl)amino-4-nitrophenylphosphorylcholine is not recommended and the assay with the fluorogenic substrate 6-hexadecanoylamino-4-methylumbel-liferylphosphorylcholine should be used with caution [54]. Assay using natural radioactive substrate is probably still the most reliable and will be described below in addition to assay using a fluorogenic substrate. 

A number of methods are available for following lipase activity. Although numerous modifications and variations have been introduced, the basic methods are (1) titration of the liberated fatty acids, (2) changes in surface tension, (3) colorimetric determination of the fatty acids, (4) use of gas-liquid chromatography, and (5) use of radioactive substrates. Kuzdzal-Savoie (1980) has reviewed the subject. 

Radioactive Substrates. Koskinen et al (1969), Luhtala et al. (1970-A,B), and Scott (1965) have used labeled triglycerides as substrates for milk lipases. This method, which is extremely sensitive, requires that the acids released by lipase action be isolated uncontaminated with any tagged glycerides. It also requires the preparation of labeled substrate and, of course, counting equipment. 

Sections of non-photosynthetic oat-tissue fed with radioactive substrates (acetate and sucrose) incorporated the label into the soluble carbohydrates and lipides, with no apparent differences between auxin-treated segments and untreated controls. 7 The tissue was apparently grown with an inadequate substrate of carbohydrate, which may explain why indole-3-acetic acid at 1 mg. per liter increased the utilization of sucrose, lipides, and organic acids by pea-stem segments in other work. None of the available investigations on cellular carbohydrates have provided very much promising information concerning the nature of the metabolic functions directly affected by the auxins. 

In eukaryotic cells the nuclear membrane separates the processes of RNA and protein synthesis. This can be demonstrated with radioactive substrates that are precursors of RNA and protein. Immediately after exposure of cells to labeled precursors, the RNA label becomes fixed in the nucleus, and the protein label becomes fixed in the cytoplasm. Eventually most of the labeled RNA becomes transferred to the cytoplasm, and a fraction of the labeled protein becomes transferred to the nucleus. 

Light enhances decarboxylation activity by proteinoids, with pyruvic acid, glucuronic, acid, glyoxalic acid, citric acid or indole-3-acetic acid as substrates 22,23). In a typical experiment, 20 mg of proteinoid is incubated with 20 pmoles of radioactive substrate for 2-3 days in 10 ml of buffer pH 4.5 (or 7.0) at 37 °C, under the irradiation of a tungsten filament bulb with a filter of 10 % CuS04 solution the COa evolved is trapped as sodium carbonate 22). 

More care is now generally given to those aspects of tracer work that have led to inconclusive results in the past, such as impurity of substrates and products and failure to establish that degradation of substrate and incorporation of smaller units was not occurring. Sources of error that are often encountered when using radioactive substrates for metabolic studies have been discussed.1... 

Use of radioactive substrates has indicated that a malted barley alpha-amylase preparation may contain transferase activity this could have a profound efiiect on the interpretation of the results shown in Table VI. However, such an effect could be due either to an enzymic impurity or to reversal of alpha-amylase hydrolytic action. It is not yet known which of these effects is occurring, and furthermore, later results have shown that the transfer activity of the plant enzymes used was so low as to have no effect on the results. ... 

Figure 6-2 shows the equilibrium distribution of radioactive S and protein in a dialysis chamber composed of two equal-volume compartments separated by a semipermeable iriembrane. Problem 6-18 illustrates the use of radioactive substrates in equilibrium binding studies. 

ASSAYS USING RADIOACTIVE SUBSTRATES 371 Compared to the original v of 62,250, we observe ... 

SPA is a newer, simpler method (Fig. 3-13b). Wc start with the same radioactive substrate, which may not necessarily need a capture group. The enzyme and potential drug are added, causing the cleavage of the substrate to some degree. Now. instead of filtering, a special resin bead coated with a... 

The enzymes requiring the largest amount of liver are carbamyl phosphate synthetase and, even more, argininosuccinate synthetase. It is therefore most desirable to develop more sensitive methods for their assay. Such methods using radioactive substrates can be devised for these enzymes. 

Another critical factor limiting the detectability in EIA is the amount of product which should accumulate before reliable measurements can be made. A fluorogenic, chemiluminescent or radioactive substrate may lower significantly the detection limit, but it may also require more sophisticated equipment. 

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