Methodology for Enzymes

Sucrose Synthase
(UDP Glucose: D-fructorse 2-a-D-glucosyl transferase E.C. 2.4.L13)

Sucrose synthase catalyses the following reversible reaction in plants.
UDP-Glucose + Fructose
Sucrose synthase
Sucrose + UDP


The synthesis activity of the enzyme is important in starch synthesis in plants. In legume nodules, however, the cleavage activity is more important as it breaksdown the major carbohydrate translocate, sucrose, into simplar forms.


Principle
The cleavage activity of sucrose synthase is easily assayed by coupling the formation of UDP-glucose to the reduction of NAD* in the presence of excess UDP-glucose dehydrogenase, and the change in A at 340 nm followed.





Materials
» HEPES-KOH buffer 0.1M (pH 7.5)
» Sucrose 0.5 M (17.11 g/100 ml)
» UDP 0.01 M (4mg/ml)
» NAD+ 0.015 M (9.95mg/ml)
» UDP-glucose dehydrogenase (0.25 mg/ml)
» Enzyme extract
   Homogenize the tissue (10g/20ml) with 10mM potassium phosphate buffer (pH 7.2) containing 1mM EDTA and 5mM 2-mercaptoethanol. Filter the homogenate through eight layers of cheesecloth and centrifuge the filtrate at 30,000g for 15 minutes, preferably at 4°C. Use the supernatant as enzyme source.


Procedure
1.  Pipette out all reagents as shown below:
Reagent
Assay (ml)
Blank (ml)
HEPES-KOH
0.2
0.3
Sucrose
0.2
0.2
UDP
0.2
0.2
Enzyme extract
0.2
0.2
UDP-glucose DH
0.1
0.1
NAD+
0.1

2.  Set the spectrophotometer to zero absorbance at 340 nm without adding NAD in the test against blank in the reference cuvette.
3.  Add NAD quickly to the test, mix well and record the initial absorbance and set a timer.
Record the decrease in A340 every minute until no further reaction is observed.


Calculation
The change in A340 is 12.0 for each micromole of UDPGper millilitre1. Express enzyme activity as micromole UDPG formed per mg of protein.





Notes
1.  The enzyme may also be assayed by following the fructose released. In such case the reaction mix will contain 20 mM HEPES-KOH, 100 mM sucrose and 2 mM UDP along with appropriate volume of enzyme. Stop the reaction after 30 min by heating in a boiling water bath for 2 min. Determine the amount of fructose released by the reducing sugar method as described earlier (p 5).


References
1.  Strominger et al (1957). Methods Enzymol3:974.
2.  Morell & Copeland (1985) Plant Physiol 78 149.