Protein Protocols Protein Protocols

Radiolabeled Prenyl Alcohols and Anaolgs 643
kinase and a geranylgeranyl phosphate kinase in eubacteria (25), together with the ability
of rat liver microsomal and peroxisomal fractions to form FPP, provide additional
evidence that these prenol pools serve as a source of lipid precursor for protein
prenylation. When proteins are labeled in this way, and are subsequently analyzed by
one-dimensional SDS-PAGE, it is possible to distinguish several major bands of
radioactivity that correspond to two apparently distinct subsets of proteins: those that
incorporate [ 3 H]-FOH and those that incorporate [ 3 H]-GGOH. But when the labeled
proteins are analyzed by high-resolution 2DE, the number of radioactive proteins that
are observed is at least fivefold greater, and three subsets of prenylated proteins can be
identified: one subset of proteins that incorporates only farnesol, a second that
incorporates only geranylgeraniol, and a third that can incorporate either prenol.
In this chapter, the advantages of using labeled prenols to dissect the differential
effects of FOH and GGOH on cellular function will be presented in the context of
studies of the role of prenylated proteins in cell-cycle progression. The ability of mammalian
cells to incorporate natural and synthetic prenol analogs (Fig. 1) into specific
proteins also will be discussed. In Subheading 4. some of the advantages and limitations
of the methods will be discussed.
2. Materials
2.1. Prenols and Analogs: Synthesis and Labeling
1. All-trans-FOH, d20 0.89 g/mL; mevalonic acid lactone; geraniol, d20 0.89 g/mL (Sigma,
St. Louis, MO).
2. Tetrahydrofarnesol was a gift from Hoffman la Roche (Basel, Switzerland). A racemic
form can be made from farnesyl acetone following the procedure for hexahydro-GGOH
(see Subheading 3.1.1.).
3. All-trans-GGOH, d20 0.89 g/mL; all-trans-GGOH [1-3H], 50–60 Ci/mmol; all-trans-FOH
[1-3H], 15–20 Ci/mmol; geraniol [1-3H], 15–20 Ci/mmol (American Radiolabeled Chemicals,
St. Louis, MO).
4. Mevalonolactone RS-[5-3H(N)], 35.00 Ci/mmol (NEN, Boston, MA).
5. Charcoal; LiAlH4 powder; MnO2; NaBH4 powder; triethyl phosphonoacetate; phosphonoacetone;
sodium ethoxide (Aldrich, Milwaukee, WI).
6. [ 3H]-NaBH4 solid, 70 Ci/mmol (Amersham, Arlington Heights, IL).
7. Silica gel 60 (F254) (Merck, Gibbstown, NJ).
8. Aquamix liquid scintillation solution (ICN Radiochemicals, Irvine, CA).
2.2. Cell Culture Reagents
1. Dulbecco’s modified Eagle media (DMEM) (glucose 4.5 g/L); penicillin (10,000 U/mL)–
streptomycin (10 mg/mL); trypsin (0.25% w/v)–1 mM ethylenediaminetetraacetate
(EDTA); nonessential amino acids (NEAA) solution 10 mM; phosphate-buffered saline
(PBS) without Ca2+ and Mg2+ ; fetal calf serum (FCS) (Gibco, Grand Island, NY).
2. Disposable culture Petri dishes (100 × 10 mm and 35 × 10 mm) (Corning Glass Works,
Corning, NY).
3. Filters, 0.22 µm (Millipore, Bedford, MA).
4. Plasma-derived, bovine serum (PDS) (Irvine Scientific, Santa Ana, CA).
5. Aquasol scintillation cocktail; thymidine [methyl-3H], 2 Ci/mmol] (NEN).
6. Trichloroacetic acid (TCA) (J. T. Baker, Phillipsburg, NJ).
7. Phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, and pepstatin A (Sigma).