| Description |
The first intrinsically fluorescent analog of geranylgeraniol, (2E,6E,8E,10E,12E,14E)-geranylgeraniol (all trans-DeltaDeltaGGOH 1) has been synthesized stereoselectively and shown to substitute for the geranylgeranyl (GG) moiety in prenyl transferase reactions and in protein-Iigand binding assays. All trans-DeltaDeltaGGOH 1 showed blue fluorescence in methanol, with lambda[ex] = 310 nm, lambda[em] = 410 nm (E 310 = 2.4 x 10 to the 4th power M-1 cm-1) and quantum yield = 0.042. It was only weakly fluorescent in aqueous solution. The prenyl transferase efficiency for all trans-DeltaDeltaGGPP 2 as a substrate for yeast protein geranylgeranyl transferase (GGTase-I) was 60% relative to GGPP. The binding of DeltaDeltaGG-AcCysMe 3 to the recombinant Rho GTPase dissociation inhibitor (RhoGDI) had a K[D] of 15.1 ± 1.2 µM, sixfold lower than the affinity of GG-AcCysMe. Incorporation of [3H]-DeltaDeltaGGOH and [3H]-GGOH into starved or lovastatin arrested NIH3T3, Glial-C6 cells and CHO-K1 has been attempted. EYFP-CVIM and EYFP-CVLL transfected CHO-K1 cells were arrested with lovastatin, then rescued with DeltaDelta;GOH and GGOH. These cell-based studies did not show that DeltaDelta;GGOH could go through salvage pathway to label small GTPases. In conclusion, DeltaDelta;GG moiety, as a novel fluorophore, has good GGTase-I substrate activity and RhoGDI binding affinity in vitro, but its applications in cell signal transduction still need more investigation. In the second part of this thesis, two antigens, succinylglycine-(geranylgeranyl)cysteine methyl ester 40 and succinylglycine-(farnesyl)cysteine methyl ester 41, were synthesized and coupled to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Immunization of rabbits produced antisera containing large amounts of polyclonal anti-geranylgeranyl and anti-farnesyl antibodies, which were purified using positive affinity column chromatography. Selective anti-farnesyl and anti-geranylgeranyl antibodies were obtained and characterized by dot blot and Western blot assays. |
