KINETICS OF PROTEIN FARNESYLTRANSFERASE - SIGMOIDAL VS HYPERBOLIC BEHAVIOR AS A FUNCTION OF ASSAY CONDITIONS

Protein farnesyl transferase (FTase) catalyzes the addition of a farne syl isoprenoid to a conserved cysteine residue in Pas and several othe r key proteins involved in cell regulation, An assay technique commonl y used to measure FTase activity involves vacuum filtration. This assa y, which traps precipitated, radiolabeled prenylated proteins on a gla ss fiber filter for analysis by scintillation counting, was designed t o be fast and accurate. In the case of FTase, substrate saturation cur ves generated by this assay technique using Pas as a substrate often s how a lag at low Pas concentrations, resulting in curves with sigmoida l character. We have found that the sigmoidal behavior is due to the u se of the filter binding assay and not to any inherent property of FTa se. Specifically, the glass fiber filters do not adequately trap preci pitated Pas proteins, especially at low concentrations. Addition of cy tosol from either bovine brain or liver, or of purified tubulin to the FTase assay mixture prior to the precipitation step, results in the a pparent formation of stable complexes of farnesylated Pas protein that can then be optimally trapped on the glass fiber filter. This appears to be at least in part due to the ability of tubulin to bind the pren yl protein reaction product. The ability to obtain accurate kinetics f or the FTase using the standard filter binding assay should greatly en hance its use to accurately assess the properties of FTase inhibitors. (C) 1996 Academic Press, Inc.