Spermidine assay

The assay was carried out in phosphate buffer with radioactive putrescine, decarboxylated S-adenosylmethionine, and enzyme. Reactions were incubated at 37°C for 90 minutes and terminated by addition of perchloric acid. The solutions were clarified by centrifugation, and the polyamines were benzoyl-ated and extracted and then analyzed. Figure 9.55 shows the analysis of samples removed at zero time (blank) and in after 60 minutes incubation (sample) at 37°C. The appearance of radioactive spermidine is shown. The rate of product formation is shown in Figure 9.56. 

Figure 9.56 Mouse brain spermidine synthetase activity as a function of time. Both the absorbance (O) and the radiometric ( ) determination of product formation represent the mean value of duplicate assays. The reproducibility was within 10%. (From Porta et al., 1981.)...

Fig. 4. Identification of ADP-ribosylated topoisomerase I following renaturation (cf. Fig. 3). The reaction mixture for the topoisomerase I assay contained in a total volume of 15 /ul 50 mAf Tris/ HCl, pH 7.5, 50 mM KCl, 10 mMMgCl, 0.5 mAf DTT, 0.1 mA/EDTA, 0.03 mg mr BSA, 0.5 Mg of form I phi X 174 DNA (BRL), and 10 m1 of renatured and diluted poly(ADPR) nonhistone proteins. ATP and spermidine were included where indicated. The incubation was carried out for 30 min at 37 C and was terminated by the addition of 2 til of proteinase K (Merck) (2 mg ml of 1% sodium dodecyl sulfate). Electrophoresis was performed for 15 h at 50 V using an 1.4% agarose gel and 1 Mg ml ethidium bromide for staining. Lane 1 control without enzyme lanes 2—4 8-, 16-, and 32-fold diluted samples lanes 5—7 8-, 16-, and 32-fold diluted samples incubated in the presence of 1 vaM ATP lanes 8-10 4-, 8-, and 16-fold diluted samples incubated in the presence of 1 mM ATP and 5 mM spermidine...

One monoamine oxidase has been found soluble in nature, and has been purified. This is the enzyme of blood serum, which has been purified 200-fold from steer plasma. The specificity of the serum enzyme is more restricted than those of the hver enzymes tryptamine and epinephrine are not oxidized rapidly, if at all, although phenylethylamine is a substrate. The polyamines spermine and spermidine are among the best substrates, and decamethylenediamine, but not shorter diamines, is also attacked. Aromatic substitutions on methylamine form substrates that permit spectrophotometric assays. Benzylamine and furfurylamine, for example, which do not absorb fight in the region used, are converted to benzaldehyde and furfuraldehyde, respectively these products have absorption maxima near 250 mja and 275 m/ . 

This scant information about aminopropyltransferases is due in part to the difficulty of measuring their activities, since one of the precursors, dSAM, is unstable and not easily available. Although its synthesis has been improved, an extinction coefficient has not been published (Samejima et al., 1978). We have developed a method for the evaluation of spermidine synthase in oat leaves (Tiburcio et al., 1986a) by a coupled reaction without using dSAM. We add SAM, [ ] putrescine, and pyridoxal phosphate to the assay mixture. Incorporation of the label into spermidine can be detected after 45 min of incubation at 37 C. Labeled spermidine is separated from labeled putrescine or spermine by elution with HCl in Dowex 50 W-H" " columns (Tiburcio et al., 1986a). A similar procedure can be used for the evaluation of spermine synthase (A. F. Tiburcio et al., unpublished observations). 

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