This chapter discusses the determination of protein prenyltransferases by using continuous fluorescence assay. In the reaction catalyzed by protein farnesyltransferase (PFTase), the farnesyl moiety of farnesyl diphosphate (FPP) is linked through a thioether bond to a cysteine four amino acid residues from the C terminus of a number of physiologically important proteins. The PFTase enzyme is a heterodimer and is purified from rat brain, bovine brain, and yeast. The assay is particularly sensitive to the type and amount of detergent used. Although 0.04% (w/v) of n-dodecyl-β-D-maltoside (DM) is satisfactory for the assays described in the chapter, alternative concentrations or detergents may be more appropriate for other peptide substrates or enzymes. To evaluate detergents for kinetic measurements, the reactions are performed at saturating concentrations of both substrates at levels of enzyme where the reaction reaches completion in a reasonable period of time. It is important to generate a high concentration of product at the end of the reaction to test the ability of the detergent to stabilize the fluorescence signal over the entire range of concentrations encountered during experiments. The evaluation of a detergent begins at a concentration near the critical micelle concentration (CMC) of the detergent.
ASJC Scopus subject areas
- Molecular Biology