α-AMYLASE ACTIVITY IN GERMINATING SEEDS
INTRODUCTION
Wheat seeds, as well as other cereal seeds, produce the enzyme α-amylase during germination.
The substrate for α-amylase is stored starch and the end product is free sugar, which is needed
for the growth of emerging embryo. Extracts from germinating grains like barley and wheat
provide a good system for studying enzyme activity. Extraction of this enzyme is accomplished
by grinding the seeds in a buffer to make a puree, then removing unwanted debris through
centrifugation. It is essential that the enzyme extract be kept cold at all points during the
extraction to prevent the action of proteases which might attack the α-amylase.
If you were to measure the level of starch in a wheat seeds, you would find that it is high in the
ungerminated seed and somewhat lower after several days of germination. The fastest rate of
disappearance of starch is found when the embryo is growing and requires the sugars that are
produced when starch is broken down. As you might suspect, the activity of α-amylase is
correlated with this loss of starch in the endosperm.
De novo synthesis of α-amylase begins in the aleurone layer of the endosperm after imbibition.
If the embryo of the seed is removed prior to inbibition, however, no increase in α-amylase
activity occurs, suggesting that a hormone or plant growth substance produced by the embryo is
necessary for the synthesis of α-amylase. The plant growth hormone gibberellic acid (GA) is the
messenger that triggers this reponse. GA is produced in the embryo and migrates to the aleurone
layer, where the synthesis of α-amylase is stimulated. The hydrolases are then secreted into the
endosperm where they cause the breakdown of reserve food, particularly starch.
In this laboratory exercise, you will examine the production of α-amylase in germinating seeds
over time. You will provide the starch in a soluble form and add the extracted enzyme. Enzyme
activity is sensitive to pH, so you can let the reaction run for a specified time, and then stop it by
lowering pH. The undigested starch may be measured spectrophotometrically after staining with
iodine. It should be noted that you are not measuring the specific activity of the enzyme (the
enzyme activity per mg total protein) in this exercise, but more likely the concentration of the
active enzyme.
When does α-amylase activity become strongest during the course of germination?
Is enzyme activity localized in the endosperm or the embryo?
PROCEDURE
Select 50 wheat seeds from each treatment and place them in a cold mortar. Obtain 40 mL of
10mM citric acid-sodium citrate buffer solution at pH 5.0 and add a small portion of the buffer to
the seeds. Keeping the mortar cold in ice, grind the seeds thoroughly, adding more fluid as you
go along until about 30 mL have been added.
Transfer your homogenate into a 50-mL labeled centrifuge tube, then use the last 10-mL of the
buffer to rinse the mortar, adding this rinse to the centrifuge tube too. The tubes should then be
centrifuged for 10 minutes at 15,000 g to remove starch grains, cell walls, mitochondria and
nuclei. Pour the supernatant of each sample into a tube and placed in an ice bucket.
For treatments 1-4, add 0.5 mL of the appropriate enzyme extract to each test tube. The reaction
will start when you add 1 mL of starch (0.1% soluble starch in 0.05 M citric acid sodium citrate
buffer at pH 5.0) to the enzyme solution. After 30 seconds, add 3.5 mL of 1 N HCl to the tubes
and mix. For treatment number 5, add HCl prior to adding the enzyme extract. For treatment
number 6, add 1 mL of water rather than starch. Use this treatment as your blank in the machine.
Add 0.5 mL of the iodine solution to the killed reaction mixture in each tube. Iodine develops a
blue color when mixed with starch; if the starch has been hydrolyzed by enzyme activity, less
blue color will be produced.
Measure the absorbance of the blue solution in a Spec 20 at 580 nm. Record your results in the
table below.
Treatment
Number
1
2
3
4
5
6
Treatment
(Germination
time)
0h
24 h
48 h
72 h
72 h
72 h
Absorbance
(A)
X-Y = Z
% Starch
Lost (100 *
Z/X)
=X
0
If X represents the amount of starch present at the beginning of the experiment (Treatment 5) and
Y represents the amount of starch remaining at the end of the experiment (separately in
treatments 1-4), then X – Y = Z, where Z is the amount of starch lost during the experiment and
the % of starch lost is 100*(Z/X).
If more than 90% of the starch disappeared in any one enzyme preparation, consider that results
to be enzyme saturated, thus comparing results of more than 90% to each other may be invalid
since the reaction may have been limited by substrate.
Plot the enzyme activity (% starch lost) as a bar graph.
QUESTIONS (Due in lecture one week from today)
When does α-amylase activity becomes strongest during the course of germination? Do your
results support the hypothesis of de novo synthesis?
Is there a lag time after imbibition of water before α-amylase appears?