Enzyme 6-phosphofructo-1-kinase is one of the most important regulatory enzymes of glycolysis. There are three phosphofructokinase genes in humans, that are diversely expressed in specific human tissue types. Enchanced expression of Pfk-L isoform, that we used for testing of inhibition upon addition of selected inhibitors, is characteristic for many human tumor types. We used Gibson assembly technology and p416GPD plasmid vector to prepare human nPFKL and sfPFKL gene, and inserted them into PFK-null yeast strain (HD114-8D). Enzyme nPfk-L was purified to homogeneity with ammonium sulfate fractional precipitation and affinity chromatography, while we purified sfPfk-L only with fractional precipitation, due to very low level of extracted sfPfk-L protein, from unstable sfPFKL yeast strain. We used both purified enzymes to test inhibition of activity, upon addition of 15 seleted inhibiotrs. We were able to determine those inhibitors, that show better inhibiton for nPfk-L activity, or sfPfk-L activity respectively. We also noticed that transformants with inserted nPFKL were quite stable, while the enzyme nPfk-L practically lost all of it`s activity after 30 min of continuous measurement. On the contrary, transformants with inserted sfPFKL were very unstable, they lost the majority of their inserted plasmids, while sfPfk-L enzyme preserved its activity.
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