-AMYLASE ACTIVITY IN GERMINATING SEEDS INTRODUCTION Wheat seeds, as well as other cereal seeds, produce the enzyme -amylase

during germination. The substrate for -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 pro ide a good system for studying enzyme acti ity. Extraction of this enzyme is accomplished by grinding the seeds in a buffer to make a puree, then remo ing unwanted debris through centrifugation. !t is essential that the enzyme extract be kept cold at all points during the extraction to pre ent the action of proteases which might attack the -amylase. !f you were to measure the le el of starch in a wheat seeds, you would find that it is high in the ungerminated seed and somewhat lower after se eral days of germination. The fastest rate of disappearance of starch is found when the embryo is growing and re"uires the sugars that are produced when starch is broken down. #s you might suspect, the acti ity of -amylase is correlated with this loss of starch in the endosperm. De novo synthesis of -amylase begins in the aleurone layer of the endosperm after imbibition. !f the embryo of the seed is remo ed prior to inbibition, howe er, no increase in -amylase acti ity occurs, suggesting that a hormone or plant growth substance produced by the embryo is necessary for the synthesis of -amylase. The plant growth hormone gibberellic acid $%#& is the messenger that triggers this reponse. %# is produced in the embryo and migrates to the aleurone layer, where the synthesis of -amylase is stimulated. The hydrolases are then secreted into the endosperm where they cause the breakdown of reser e food, particularly starch. !n this laboratory exercise, you will examine the production of -amylase in germinating seeds o er time. 'ou will pro ide the starch in a soluble form and add the extracted enzyme. Enzyme acti ity is sensiti e to p(, so you can let the reaction run for a specified time, and then stop it by lowering p(. The undigested starch may be measured spectrophotometrically after staining with iodine. !t should be noted that you are not measuring the specific acti ity of the enzyme $the enzyme acti ity per mg total protein& in this exercise, but more likely the concentration of the acti e enzyme. When does -amylase acti ity become strongest during the course of germination) !s enzyme acti ity localized in the endosperm or the embryo) PROCEDURE *elect +, wheat seeds from each treatment and place them in a cold mortar. -btain ., m/ of 0,m1 citric acid-sodium citrate buffer solution at p( +., and add a small portion of the buffer to the seeds. 2eeping the mortar cold in ice, grind the seeds thoroughly, adding more fluid as you go along until about 3, m/ ha e been added.

Transfer your homogenate into a +,-m/ labeled centrifuge tube, then use the last 0,-m/ of the buffer to rinse the mortar, adding this rinse to the centrifuge tube too. The tubes should then be centrifuged for 0, minutes at 0+,,,, g to remo e starch grains, cell walls, mitochondria and nuclei. 4our the supernatant of each sample into a tube and placed in an ice bucket. 5or treatments 0-., add ,.+ m/ of the appropriate enzyme extract to each test tube. The reaction will start when you add 0 m/ of starch $,.06 soluble starch in ,.,+ 1 citric acid sodium citrate buffer at p( +.,& to the enzyme solution. #fter 3, seconds, add 3.+ m/ of 0 7 (8l to the tubes and mix. 5or treatment number +, add (8l prior to adding the enzyme extract. 5or treatment number 9, add 0 m/ of water rather than starch. :se this treatment as your blank in the machine. #dd ,.+ m/ of the iodine solution to the killed reaction mixture in each tube. !odine de elops a blue color when mixed with starch; if the starch has been hydrolyzed by enzyme acti ity, less blue color will be produced. 1easure the absorbance of the blue solution in a *pec <, at +=, nm. >ecord your results in the table below. Treatment 7umber 0 < 3 . + 9 Treatment $%ermination time& ,h <. h .= h D< h D< h D< h #bsorbance $#& ?-' @ A 6 *tarch /ost $0,, B AC?&

@? ,

!f ? represents the amount of starch present at the beginning of the experiment $Treatment +& and ' represents the amount of starch remaining at the end of the experiment $separately in treatments 0-.&, then ? E ' @ A, where A is the amount of starch lost during the experiment and the 6 of starch lost is 0,,B$AC?&. !f more than F,6 of the starch disappeared in any one enzyme preparation, consider that results to be enzyme saturated, thus comparing results of more than F,6 to each other may be in alid since the reaction may ha e been limited by substrate. 4lot the enzyme acti ity $6 starch lost& as a bar graph. QUESTIONS (Due in lecture one ee! "ro# to$%&' When does -amylase acti ity becomes strongest during the course of germination) Go your results support the hypothesis of de novo synthesis) !s there a lag time after imbibition of water before -amylase appears)