Assays Using Radioactive Substrates

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

The authors of this review have used I-human serum albumin for determining proteolytic activity in various types of foodstuffs, enzymatic preparations (determination of the contaminating proteolytic activity), pharmaceuticals and other materials Proteolytic activity assays using radioactively labeled substrates... 

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. 

Clinical studies have considered the activity of diamine oxidases to be a useful parameter (Baylin, 1977 Luk etai, 1980) thus, many different assays to determine the activity of diamine oxidases have been proposed. Procedures using radioactive substrate (Okuyama and Kobayashi, 1961 Kusche et ai, 1973) achieve high sensitivity and most recently the use of high-performance liquid chromatography has been described (Biondi et ai, 1984). 

The present HG-PRT micro assay introduces radioactive substrates to the PMC-technique. Therefore, is becomes possible with the PMC to use natural substrates with high specificity and sensitivity. Moreover radioactive substrates and products can be separated in a straightforward way to check possible side reactions. 

Mnk assay (using S6peptide as substrate) Each reaction contains 20 1 of immunoprecipitated or purified recombinant Mnk, as described previously, and S6 peptide (KEAKEKRQEQIAKKRRLSSLRASTSKSESSQK, final concentration = 200 pM) in kinase buffer. To initiate the reaction, 6 p of the following mix is added 5 pi 5x kinase buffer, 0.25 pi 10 mM ATP, 1 pCi [7 32P]ATP, and H20. Reactions are carried out in an orbital shaker at 30° and 900 rpm, for example, for 20 min (Mnkl) or for 5 min (Mnk2) and stopped by spotting 20 pi of the reaction mixture onto P81 paper. The filters are washed in 1% orthophosphoric acid three times. The radioactivity is determined by using a liquid scintillation counter. 

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. 

This enzyme is deficient in Niemann-Pick disease type A/ (Table 4.4.1, Fig. 4.4.1). The assay with radioactive natural substrate is based on the method described by Wenger [59]. The assay with fluorescent substrate is based on the method described by van Diggelen et al. [54]. These assays have not yet been validated in the authors laboratory for use with dried blood spots. For this application the reader is referred to Chamoles et al. [9]. 

A more explicit demonstration of chain-shortening was achieved by performing assays using 3-14C-hexadecanoic acid as substrate (IL) Over 80% of the radioactivity in the isolated chain-shortened tetradecanoic acid was in the 1-position, showing that most of the reaction was direct, though a small amount may have undergone degradation/resynthesis. 

The separation of the substrate and the product was accomplished by reversed-phase HPLC on an ODS column. The column was eluted isocratically with a mobile phase of methanol-water (33 67 v/v). The compounds were detected at 254 nm. The separations obtained are shown in Figure 9.121. Radiolabeled substrates were also used, and the eluent was assayed for radioactivity on fractions collected during the elution. 

Myosin light chain kinase is a Ser/Thr-type protein kinase involved in regulation of smooth muscle. The enzyme is also found in smooth muscle and platelets. This assay uses a synthetic substrate that is not radioactively labeled. 

A radiometric and a spectrometric assay have been developed to measure PhaC activity. The radiometric assay measures the incorporation of isotope-labeled hydroxyacyl moieties into the polyester, which is present from the beginning as primer [17]. [3-14]ft-(-)-3-hydroxybutyryl-CoA or [3H]-P,S-3-hydroxybutyryl-CoA or in principle any other CoA thioester of a radioactively-labeled hydroxyacyl moiety could be used as substrate. Only the radioactivity that is really incorporated into the insoluble polyester is measured. The time course of the assay, the need to synthesize the substrates and the high costs make the assay very inconvenient and it is hardly used anymore. A more convenient assay is the spectrometric assay which measures the release of coenzyme A during the polymerization reaction in presence of Ellmann s reagent 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) yielding 5 -thio(2-nitrobenzoate) that absorbs at about 412 nm [16], Here, the enzyme activity is measured directly without delay. However, it is not the formation of the polymeric product that is measured, but the release of coenzyme A, which can also be due to the hydrolytic cleavage of the substrate by another enzyme that does not have any PhaC activity, like a thioesterase. Nevertheless, this assay is now most frequently used due to its convenience. 

Various assays are used to detect and quantify proteins. Some assays use a light-producing reaction or radioactivity to generate a signal. Other assays produce an amplified colored signal with enzymes and chromogenic substrates. 

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