Protein Protocols Protein Protocols

Radiolabeled Prenyl Alcohols and Anaolgs 653
Fig. 7. (continued) (B) Whole-cell lysate from [ 3 H]-GGOH-labeled cells that contained
475 µg protein and 390 × 10 3 cpm. Film was exposed for 10 wk. The major proteins that
incorporated both FOH and GGOH are circled. At least nine additional clusters of minor
proteins display a similar ability to incorporate either prenol, but have not been circled
(see also Note 7). The position of the internal standard, [ 3 H]-NEM-labeled soybean trypsin
inhibitor (6250 cpm per gel; see Subheading 3.3.3.), is indicated by a square.
radioactivity are seen in the 21–28 kDa range, corresponding to Ras and Ras-related small
GTPases (2,3). In contrast, only a few protein bands are visible when simvastatin is omitted
from the incubation.
3. It also is necessary to block endogenous MVA synthesis with simvastatin, for efficient
incorporation of [ 3 H]-FOH into specific cellular proteins. However, this is not the case for
[ 3 H]-GGOH labeling, which proceeds equally well in the presence or absence of
simvastatin (Fig. 5).
4. In investigations of the cellular uptake of MVA and its derivatives, the authors typically
found uptake of less than 0.1% of the added [ 3 H]-MVA, 2% of the added [ 3 H]-FOH, and
more than 10–15% of the added [ 3 H]-GGOH. The efficient uptake of GGOH by cells
permits rapid labeling of geranylgeranylated proteins. Protein bands from one-dimensional
SDS-PAGE are detectable after labeling the cells for only 1–2 h, and subsequent gels
require only a few days of film exposure.
5. Both [ 3 H]-GGOH labeling of proteins and the GGOH-mediated prevention of simvastatininduced
inhibition of DNA synthesis show parallel dose-response sensitivities to a similar
range of GGOH concentrations (up to 2.5 µM; Fig. 4).
6. The labeling approach described here can also be utilized for investigating natural or
synthetic isoprenoid analogs (Fig. 6). When cells were treated with [ 3 H]-2-cis-GGOH
(precursor of dolichols), proteins are only weakly labeled, compared to all trans-GGOH.
Under parallel conditions, the addition of unlabeled 2-cis-GGOH failed to prevent the
inhibitory effect of simvastatin on DNA synthesis (Table 1).