Polygalacturonase assay
This enzyme is famous for being involved in the development of the GMO tomatoes (more information from the link at the foot of this page). The cells of these tomatoes have been genetically modified to contain a reversed copy of the gene for this enzyme. This produces anti-sense mRNA which combines with the normal mRNA for polygalacturonase, effectively supressing its synthesis. As a result the GM tomatoes have only 1% of usual polygalacturonase activity, and soften much more slowly than unmodified tomatoes - thus extending their shelf-life.
The normal role of polygalacturonase is to hydrolyse pectins during fruit ripening, which leads to softening of the fruit. The plant hormone ethene has been shown to promote the translation of polygalacturonase mRNA, raising the levels of the enzyme in ripening fruit.
Some plant pathogenic fungi, such as Phytophthora infestans (potato blight), secrete polygalacturonase during their attack of host plant cell walls.
Another interesting fact is that the regulation of the production of the enzyme is partly due to ethene (ethylene), which induces the translation of polygalacturonase mRNA, rather than regulating thetranscription step.
The principle of this assay is to mix an extract of fruit (or vegetable) with a viscous solution of polygalacturonic acid, and then note the decrease in viscosity due to its breakdown by the enzyme. |
Make the extract of the fruit (or vegetable) in a 40 mM sodium acetate solution (1), buffered to pH 5.0 with hydrochloric acid. Use a volume of buffer equivalent to the mass of the fruit or vegetable sample (e.g. 10 g fruit + 10 cm3 buffer). Grind the sample in a mortar and pestle, or if larger quantities are required, do this in a blender.
To obtain a clear enzyme extract from the mashed up fruit, then either filter it (could be speeded up on a Buchner funnel) or centrifuge to produce a clear supernatant containing the enzymes (faster and simpler).
Then add an equal volume of this enzyme extract / supernatant to the polygalacturonic acid solution (2). Set up a control by adding an equal volume of sodium acetate to the same volume of another polygalacturonic acid sample.
Incubate these mixtures in a water bath at 40°C. Measure the viscosity at the start and after incubation for about one hour (see next paragraph).
Measure the initial vicosity of these mixtures by drawing up 1 cm3 into a glass pipette, and then time how long it takes the mixture to drain out under gravity, to the 0.9 cm3 mark. Repeat the measurement three times and calculate a mean time. Re-measure the viscosity after incubation (at 40°C, for 1 hour).
Because of variations in the diameter etc. of pipettes, it would be best to use the same pipette for all readings, carefully rinsing it out between them. Make sure the pipette is held vertically, possibly with a retort stand and clamp.
Preliminary experiments with a ripe tomato have shown a 50% decrease in viscosity after incubation for 1 hour.
(1) Sodium acetate buffer - 40 mM solution made up, then add 1 M HCl dropwise, whilst pH is monitored with a pH meter.
(2) Make up a 3.2 % (w/v) of polygalacturonic acid (sodium salt), by mixing the powder with warm distilled water, stirring it thoroughly with a glass rod and then placing the mixture in a boiling water bath for 10 minutes - stirring occasionally. Then filter the warm solution or centrifuge it to provide a clear, viscous solution of polygalacturonic acid for these experiments.
