TY - JOUR
T1 - Continuous Fluorescence Assay for Protein Prenyltransferases
AU - Cassidy, Pamela B.
AU - Dolence, Julia M.
AU - Dale Poulter, C.
N1 - Funding Information:
I thank Professor Yoel Kloog and Professor Michael Gelb for critical reading of the manuscript and for helpful suggestions. Y. R. is a recipient of the Aflon Fellowship Award by the Council for Higher Education of Israel.
Funding Information:
Dansylated peptides were synthesized and purified by Dr. R. Schackmann, Utah Regional Cancer Center Protein/DNA Core Facility (Salt Lake City, UT). J.M.D. is a National Institutes of Health Postdoctoral Fellow, National Institute of General Medical Sciences, GM-15286. This work was supported by NIH Grant GM-21328.
PY - 1995
Y1 - 1995
N2 - 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.
AB - 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.
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U2 - 10.1016/0076-6879(95)50060-X
DO - 10.1016/0076-6879(95)50060-X
M3 - Article
C2 - 7651159
AN - SCOPUS:0029028589
VL - 250
SP - 30
EP - 43
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
IS - C
ER -