Didehydrogeranylgeraniol(DeltaDeltaGGOH) and anti -isoprenyl antibodies for protein prenylation.

摘要

The first intrinsically fluorescent analog of geranylgeraniol, (2 E,6E,8E,10E,12 E,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 lambdaex = 310 nm, lambda em = 410 nm (epsilon310 = 2.4 x 104 M-1cm-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 muM, 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 DeltaDeltaGGOH and GGOH. These cell-based studies did not show that DeltaDeltaGGOH could go through salvage pathway to label small GTPases. In conclusion, DeltaDeltaGG 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.