ABSTRACT
New fluorescent analogues of farnesol and geranylgeraniol have been prepared and then converted to the corresponding pyrophosphates. These analogues incorporate anthranylate or dansyl-like groups anchored to the terpenoid skeleton through amine bonds that would be expected to be relatively stable to metabolism. After addition of the alcohols or the pyrophosphates to the culture medium, their fluorescence is readily observed inside a human-derived leukemia cell line. Enzyme assays have revealed that the farnesyl pyrophosphate analogue is an inhibitor of FTase, while the corresponding alcohol is not. These results, together with Western blot analyses of cell lysates, indicate that the farnesyl pyrophosphate analogue penetrates the cells as an intact pyrophosphate and that it does so at a biologically relevant concentration.
Subject(s)
Fluorescence , Polyisoprenyl Phosphates/chemical synthesis , Polyisoprenyl Phosphates/pharmacokinetics , Cell Line, Tumor , Cells, Cultured , Humans , Molecular Structure , Polyisoprenyl Phosphates/chemistry , Protein Prenylation/drug effects , Sesquiterpenes , ras Proteins/drug effects , ras Proteins/metabolismABSTRACT
Protein prenylation is a posttranslational lipid modification in which C(15) and C(20) isoprenoid units are linked to specific protein-derived cysteine residues through a thioether linkage. This process is catalyzed by a class of enzymes called prenyltransferases that are being intensively studied due to the finding that Ras protein is farnesylated coupled with the observation that mutant forms of Ras are implicated in a variety of human cancers. Inhibition of this posttranslational modification may serve as a possible cancer chemotherapy. Here, the syntheses of two new farnesyl diphosphate (FPP) analogues containing photoactive benzophenone groups are described. Each of these compounds was prepared in six steps from dimethylallyl alcohol. Substrate studies, inhibition kinetics, photoinactivation studies, and photolabeling experiments are also included; these experiments were performed with a number of protein prenyltransferases from different sources. A X-ray crystal structure of one of these analogues bound to rat farnesyltransferase illustrates that they are good substrate mimics. Of particular importance, these new analogues can be enzymatically incorporated into Ras-based peptide substrates allowing the preparation of molecules with photoactive isoprenoids that may serve as valuable probes for the study of prenylation function. Photoaffinity labeling of human protein geranylgeranyltransferase with (32)P-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from the beta-subunit of this enzyme. These results clearly demonstrate the utility of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP or GGPP as their substrates.
Subject(s)
Dimethylallyltranstransferase/antagonists & inhibitors , Polyisoprenyl Phosphates/chemical synthesis , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Benzophenones , Crystallography, X-Ray , Dimethylallyltranstransferase/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Ethers , Humans , Inhibitory Concentration 50 , Models, Molecular , Phosphorus Radioisotopes , Photochemistry , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/pharmacokinetics , Rats , Sesquiterpenes , Structure-Activity Relationship , YeastsABSTRACT
Squalene synthase catalyses the synthesis of squalene from trans-farnesyl diphosphate in 2 separate steps requiring NAD(P)H. The kinetics of this enzyme in different fractions extracted from a wild-type Saccharomyces cerevisiae were studied. Although this protein is known to be a membrane-bound enzyme, we have found a cytosolic squalene synthase activity besides the microsomal enzyme. A spectrophotometric enzyme assay, not involving isotopic labelling, was established. The relative synthesis of presqualene and squalene was evaluated by using different substrate and cofactor concentrations during the incubation. The involvement of a single catalytic site promoting the 2 reactions of squalene synthesis is suggested.
Subject(s)
Farnesyl-Diphosphate Farnesyltransferase/pharmacokinetics , Saccharomyces cerevisiae/enzymology , Squalene/chemistry , Cytosol/enzymology , In Vitro Techniques , Microsomes/enzymology , Polyisoprenyl Phosphates/pharmacokinetics , Sesquiterpenes , Spectrophotometry , Substrate SpecificityABSTRACT
The current-voltage steady-state characteristics, cyclic voltammograms and capacitance-voltage steady-state relationships of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl-12 phosphate have been studied. Sustained fluctuations of the capacitance of dolichyl phosphate modified bilayers under applied voltage were observed. The results suggest that the dynamics of dolichyl phosphate molecules in membranes can be regulated by transmembrane electrical potential.
