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5.1.1 Specific Incorporation

If intact plants or animals are used, incorporation of a labelled intermediate can be extremely low (0.01 to 1%) because many processes and organs are competing for the labelled compound. The specific incorporation of the isotope is defined as the specific activity of the desired product divided by the specific activity of the starting material expressed as a percentage. It is an important measure in biosynthesis studies. The specific activity of a labelled compound is in turn defined as the amount of radioactivity in a given amount of material (expressed as counts per minute per mole (cpm mol-1) or cpm g-1 or disintegrations per minute, dpm). Radioactivity is generally expressed in becquerels (1 Bq = 1 disintegration sec-1) or in curies (1 Ci = 3.7 x 1010 Bq).

A scale of label incorporation attributed to Sir Derek Barton says that 1% is excellent, 0.1% is good, 0.01% is positive and 0.001% or less is dubious, probably negative. Especially using 13C (which is followed by NMR spectroscopy) it is necessary to obtain at least 1% incorporation for successful deduction of the biosynthesis. Better incorporation of the intermediate can be achieved with micro-organisms, tissue culture of insect cells, or excised glands (see coccinellines, Chapter 3), or by having a cell-free system of partially purified enzymes. Alternatively, now genes for making certain enzymes can be inserted into microorganisms (bacteria or yeasts) to carry out the biosynthesis more conveniently. This will become increasingly useful as more genes are sequenced.

5.1.2 Locating the Site of Synthesis

(Z)-10-Heptadecen-2 -one is the major component of the aggregation pheromone of the fruit fly Drosophila buzzatii and 2-tridecanone from the same insect has an inhibiting effect on the aggregation. To find where and how the pheromone was synthesized, insects were separated into heads, thoraces and abdomens (Skiba and Jackson, Insect Biochemistry and Molecular Biology, 1993, 23, 375). Each of these body parts was incubated with sodium [l-14C]acetate. No radioactive pheromone was recovered from the heads or thoraces, but 1.1% of the recovered label was incorporated into pheromone by the abdomens. In addition, 0.2% of label was incorporated into 2-tridecanone (Figure 5.1). Since the site had been traced to the abdomen, in a second series of experiments they separated various parts of the abdomen and found only the ejaculatory bulb (part of the genital organs) of mature male flies synthesized the compounds. This time 3.3% of the recovered label (0.4% of the applied label) was recovered in the heptadecenone, and 1.0% of the recovered label (0.1% of the applied label) in tridecanone.

Figure 5.1 The compounds studied by radio-labelling, which are synthesized in Drosophila buzzatii

Figure 5.1 The compounds studied by radio-labelling, which are synthesized in Drosophila buzzatii

As an example of one experiment, 0.2 |j,l of sodium [l-14C]acetate containing 440,000 cpm (counts per minute) was incubated with the ejaculatory bulbs at pH 6.8 in phosphate buffer. The products were extracted with hexane and subjected to radio-gas chromatography. The total label recovered was 54,000 cpm, but most of this was in fatty acids, 1162 cpm were recovered in the heptadecenone peak (2.15% of recovered label, 0.26% of applied label) and 625 cpm in tridecanone (1.15% of recovered label, 0.14% of applied label). The experiments effectively proved the site of biosynthesis and showed the compounds were being synthesized from acetate units.

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