You are purifying the enzyme Hexokinase which converts Glc r

You are purifying the enzyme Hexokinase (which converts Glc rightarrow Glc-6-PO_4) from rat liver cells. The figure shows your results from the various purification steps. Both the SDS-PAGE results and the corresponding Western blot (using a Hexokinase-specific antibody) is shown. Galactose is a competitive inhibitor of Hexokinase. Based on these results, answer the questions that follow: Can we conclude that we have purified Hexokinase? What evidence supports your answer? We can conclude that most likely, the sample in the Galaxies affinity column lane will have the highest specific activity. This conclusion is based on what data shown? Which lane is most likely to show the lowest activation energy for the Glc rightarrow Glc-6-PO_4 reaction catalyzed by Hexokinase? I measure the K_m and V_max for Hexokinase in the Total cell lysate and the Galactose column purified sample, using the same volume of each sample. MOST LIKELY, will they be different in the two samples? Explain your answer.

Solution

a) Yes, since one band after the Galactose column is there in both SDS-PAGE and Western blot.
b) The thickness of the band in the Western blot shows the same.
c) Since Galactose inhibits the reaction, all the lanes ecept the Galactose affinity purified enzyme will have the same
activation energy.
d) Since galactose is the competitive inhibitor of hexokinase, the reaction wil exhibit normal V_max but increased K_m.