Dihydrofolate reductases assay

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

Remy 1, Campbell-Valois FX, Michnick SW. Detection of protein-protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase. Nat. Protoc. 2007 2 2120-2125. 

Campbell-Valois FX, Michnick SW. Synthesis of degenerated libraries of the ras-binding domain of raf and rapid selection of fast-folding and stable clones with the dihydrofolate reductase protein fragment complementation assay. Methods Mol. Biol. 2007 352 249-274. 

Enzyme Assays Activities of thymidylate synthase (Rode et al. 1990), thymidine kinase (Tsukamoto et al. 1991) and dihydrofolate reductase (Mathews et al. 1963) were assayed according to previously published procedures. dUTP-ase activity was determined by coupling with the thymidylate synthase-catalyzed reaction and measuring tritium released from [ H]dUMP (Golos and Rode 1999). The activity unit was defined as the enzyme amount required to convert 1 pmol of substrate per 1 min at 37°C. 

For the assay of dihydrofolate reductases (see p 612) suspend 66.5mg of DHFA in lOmL of O.OOIM HCl containing lOmM dithiothreitol (DTT stock made from 154mg in lOmL H2O making O.IM), shake well and freeze in 400 total volume of 2mL) to give a clear solution. To estimate the concentration of DHFA in this solution, dilute... 

R.J. Ha3mian, R. McGready and M.B. Van Der Weyden. A rapid radiometric assay for dihydrofolate reductase. Anal. Biochem. 

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

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