Kinetics of protein farnesyltransferase: sigmoidal vs hyperbolic behavior as a function of assay conditions.
Protein farnesyl transferase (FTase) catalyzes the addition of a farnesyl isoprenoid to a conserved cysteine residue in Ras and several other key proteins involved in cell regulation. An assay technique commonly used to measure FTase activity involves vacuum filtration. This assay, which traps precipitated, radiolabeled prenylated proteins on a glass fiber filter for analysis by scintillation counting, was designed to be fast and accurate. In the case of FTase, substrate saturation curves generated by this assay technique using Ras as a substrate often show a lag at low Ras concentrations, resulting in curves with sigmoidal character. We have found that the sigmoidal behavior is due to the use of the filter binding assay and not to any inherent property of FTase. Specifically, the glass fiber filters do not adequately trap precipitated Ras proteins, especially at low concentrations. Addition of cytosol from either bovine brain or liver, or of purified tubulin to the FTase assay mixture prior to the precipitation step, results in the apparent formation of stable complexes of farnesylated Ras 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 prenyl protein reaction product. The ability to obtain accurate kinetics for the FTase using the standard filter binding assay should greatly enhance its use to accurately assess the properties of FTase inhibitors.
Thissen
J A
JA
Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686, USA.
Casey
P J
PJ
D019883:Alkyl and Aryl Transferases; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D007700:Kinetics; D014166:Transferases / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
D014404:Tubulin; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase