Structural Biochemistry/Micro-putification

PFK-1

Phosphofructokinase-1 (PFK-1) is a putative controlling enzyme in glycolysis. Cancer cells have defective mitochondria, which forces them to depend on glycolysis as their main source of energy. Ascorbate (commonly known as Vitamin C) has been shown to inhibit PFK-1 and Lactate Dehydrogenase (LDH). In the muscle cells, when the muscle is active, PFK-1 and LDH form a complex with contractile proteins in the muscle, protecting them from inhibition allowing for glycolysis (glucose is degraded and ATP is made. When the muscle is at rest, it is hypothesized that the complex is not formed, and PFK-1 and LDH are inhibited by ascorbate, glycolysis does not occur and glucose is stored as glycogen. Glycogen storage mainly occurs in the liver and muscle tissue. It is also hypothesized that because cancer cells depend mainly on glycolysis for energy, that PFK-1 in cancer cells will not be inhibited by ascorbate. In order to test this hypothesis it is important to be able to purify PFK-1. Because aldolase (Ald) protects from inhibition, the purified PFK-1 must not have aldolase in it in order to properly test the hypothesis.

Micro-purification Method

Rabbit muscle was used as a source of PFK-1. Preparation of three assays (PFK-1 assay, LDH assay, and aldolase assay) were necessary to test the enzyme activity after each step using a spectrophotometer. This is done to keep track of what is removed and what stays after each step.

Preparation
Rabbit muscle was blended in a solution of ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT, and sodium fluoride (NaF), then centrifuged and washed in order to remove soluble proteins. This was done multiple times to try and remove some LDH and aldolase.

Heat Step The pellet containing the PFK-1 was then resuspended in a solution of Tris, MgSO4, EDTA, ATP, and DTT and then submerged in a heat bath and then placed in an ice bath. This was done to denature the other proteins while solubilizing and keeping safe the PFK-1 by using ATP and magnesium. The suspension was centrifuged and the supernatant saved.

Ion-exchange and Dye Columns
The supernatant was then placed on four different columns, all standardized with 40TP8 and DTT. The four columns were: blue agarose, brown agarose, blue dextran, and DEAE-Sephacel. The supernatant was allowed to drip through the columns by gravity and then collected to be tested. Theoretically, the PFK-1 should be stuck to the columns at that point. Solutions of 40TP8/ DTT were then added to the columns and collected in fractions. And then solutions containing an increased concentration of TP8 were added and then collected in fractions. The purpose of adding increasing concentrations of TP8 was to wash off any other proteins with a weaker affinity for the beads in the columns first. An additional method using a vacuum filtration system was tried, but would pull the PFK-1 and aldolase through the column too quickly, not giving PFK-1 enough time to stick to the column, making this method counterproductive.

Gel Electrophoresis Samples taken from the column were then tested for purity by polyacrylamide gel electrophoresis (PAGE)

Results and Discussion

The DEAE Sephacel column seemed to have the highest yield of PFK-1 activity out of the four columns, but when tested using PAGE there was a lot of contamination. As mentioned before, there shouldn’t be aldolase because it inhibits PFK-1, which would make testing the affects of ascorbate on PFK-1 very hard to do. The blue agarose, while it didn’t have as high of a yield as DEAE Sephacel, showed to be very pure when tested by PAGE. The brown agarose showed potential at producing high yields of PFK-1, but after continuous testing, while it was good at removing aldolase, the percent yield of PFK-1 was too low.
Blue agarose so far has shown to be the best choice out of the four, but continuous testing/adjusting must be done before using this process on cancerous tissue. This is because while blue agarose removes aldolase well, the yield is still not high enough for it to be efficient to use with cancer tissue.

References

1. Queja, A., Marshall, A., Willaims, A., & Russell P.J. (2012, June). Micro-purification of Rabbit Phosphofructokinase-1. Poster presented at the biennial symposium of the Intercultural Cancer Council (ICC), Houston, TX.

2. Russell P, Williams A, Marquez K, Tahir Z, Hossein B, Lam K. 2008. Some characteristics of rabbit muscle phosphofructokinase-1 inhibition by ascorbate. J Enzyme Inhib & Med Chem; 23: 411-417

3. Vassault, A. 1983. Methods of Enzymatic Analysis, Enzymes I: Oxidoreductases, Transferases Vol. III, Verlag Chemie, Basel, pp. 118–126.