Spermidine Synthetase Porta et al 1981

The enzyme spermidine synthetase catalyzes the conversion of putrescine to spermidine. The separation of the substrate from the product was accomplished on a reversed phase column (Ci8) using a mobile phase of 60% methanol (v/v). The compounds in the eluent were detected by scintillation counting or by UV at 254 nm as shown in Figure 9.101 (labeled Blank).

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 9.55 HPLC separation of the benzoyl derivatives of putrescine (PT) and spermidine (SPD) contained in a standard reaction sample and in the corresponding blank after 60 minutes of incubation at 37°C. Fractions (0.8 mL) were collected for determination of radioactivity. The arrows indicate a change in detector sensitivity from 0.2 to 0.05 absorbance unit full scale. (From Porta et al., 1981.)

Figure 9.55 HPLC separation of the benzoyl derivatives of putrescine (PT) and spermidine (SPD) contained in a standard reaction sample and in the corresponding blank after 60 minutes of incubation at 37°C. Fractions (0.8 mL) were collected for determination of radioactivity. The arrows indicate a change in detector sensitivity from 0.2 to 0.05 absorbance unit full scale. (From Porta et al., 1981.)

The enzyme was prepared from mouse brain homogenized in phosphate buffer. An S-100 solution was prepared and used as the source of the synthetase.

It is of interest to note the use of the "sensitivity shift" procedures, illustrated in Figure 9.55. Arrows on the figure indicate where detector sensitivity was increased to allow for the detection of low levels of the product.

0 30 60 90 120

Incubation time, min

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.)

0 30 60 90 120

Incubation time, min

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.)

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