WHOLE CELL RUBISCO ENZYME ASSAY

(Tabita, Caruso and Whitman 1978)

Because of high variability in cell extraction it is preferable to do whole cell assays. This procedure can be adapted to diel studies. It is best to use exponentially growing cultures.

1. When in doubt, keep cells on ice – have an ice bucket ready.
2. This is a radio-assay. The amount of 14C used must be logged in the isotope book.

1. MOPS-KOH (100 mM MOPS-KOH pH7.5, 10 mM EDTA) .
2. Buffer A  (100 mM MOPS-KOH pH7.5).
3. Buffer B  (50 mM NaHCO3, 25 mM Mg Acetate, sterile filter).
4. Hot Buffer B  Add 100 μl Na14CO3 (2 mCi/ml) to 10 ml Buffer B.
5. 8 mM RuBP pH 6-6.8 (aliquot and freeze)

1. Harvest 20 ml of cells in a sterile oak-ridge tube. Do this by spinning at 15,000 g for 15 minutes (refer to table for correct speed).
2. Dump supernatant and wash cells with 10 ml of sterile MOPS-KOH buffer.
3. Re-spin and dump the supernatant.
4. Resuspend the pellet in 0.5 ml MOPS-KOH buffer and transfer to microfuge tube.
5. Add 0.25 ml tolune.
6. Stir gently for 3 minutes and place on ice for 10 minutes.
7. Aspirate out the toluene. Aliquot into 150 μl aliquots into sterile 0,5 ml tubes and freeze or use immediately in assay. If followed by assay keep aliquots on ice.

1. Split one 150 μl aliquot three ways into 50 μl each.
2. Add 75 μl Buffer A to each tube.
3. Incubate at 30˚ C for 5 minutes.
4. Add 100 μl HOT Buffer B to each tube as quickly as possible.
5. Incubate at 30˚ C for 5 minutes.
6. Add 25 μl RuBP to two of the three aliquots and 25 μl DI to the third.
7. Incubate one of the tubes containing RuBP and the tube containing DI for 30 minutes at 30˚ C and terminate the reaction with 100 μl propionic acid.
8. Do not incubate the second tube containing RuBP and terminate the reaction immediately using propionic acid.
9. Let tubes outgas for 60 minutes while vortexing every 15.
10. Add 200 μl to 10 ml of Ecoscint A in a scintillation vial and count.

1. In triplicate add 10 μl Hot Buffer B to 1 ml cold Buffer B in a microfuge tube and vortex.
2. Add 100 μl of this solution to 10 ml Ecoscint A in a scintillation vial and count immediately.
3. Calculate the cpm/nmol HCO3-. This will enable you to convert CPMs into nM HCO3- fixed. There are 50 nM of HCO3- in the 100 μl aliquot that was counted for the control sample in the case, where the concentration of cold HCO3- is 20 mM. If the control counts are 30,000 then there are 30,000 cpm/50 nM or 500 cpm/nM. This changes from experiment to experiment and needs to be determined.