carbocyanines and parinaric-acid

Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his-tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally occurring fluorescent cis-parinaroyl-CoA with very high affinity (K(d)=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his-tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor-4alpha (HNF-4alpha), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4alpha were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4alpha (intermolecular distance of 73 A) at high affinity (K(d)=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP.

Topics: Acyl Coenzyme A; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Carbocyanines; Circular Dichroism; Cloning, Molecular; Coenzyme A Ligases; Diazepam Binding Inhibitor; Escherichia coli; Fatty Acids, Unsaturated; Fluorescence Resonance Energy Transfer; Glycerol-3-Phosphate O-Acyltransferase; Hepatocyte Nuclear Factor 4; Histidine; Mice; Molecular Sequence Data; Recombinant Proteins; Spectrophotometry, Ultraviolet

Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost nothing is known regarding the structural properties, cholesterol dynamics or intracellular factors affecting caveolar cholesterol dynamics. A non-detergent method was employed to isolate caveolae/raft domains from purified plasma membranes of murine fibroblasts. A series of fluorescent lipid probe molecules or a fluorescent cholesterol analogue, dehydroergosterol, were then incorporated into the caveolae/raft domains to show that: (i) fluorescence polarization of the multiple probe molecules [diphenylhexatriene analogues, DiI18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), parinaric acids and NBD-stearic acid [12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid] indicated that acyl chains in caveolae/raft domains were significantly less 'fluid' (i.e. more rigid) and the transbilayer 'fluidity gradient' was 4.4-fold greater than in plasma membranes; (ii) although sterol was more ordered in caveolae/raft domains than plasma membranes, spontaneous sterol transfer from caveolae/raft domains was faster (initial rate, 32%; half-time, t(1/2), 57%) than from the plasma membrane; (iii) although kinetic analysis showed similar proportions of exchangeable and non-exchangeable sterol pools in caveolae/raft domains and plasma membranes, addition of SCP-2 (sterol carrier protein-2) 1.3-fold more selectively increased sterol transfer from caveolae/raft domains by decreasing the t(1/2) (50%) and increasing the initial rate (5-fold); (iv) SCP-2 was also 2-fold more selective in decreasing the amount of non-exchangeable sterol in caveolae/raft domains compared with plasma membranes, such that nearly 80% of caveolar/raft sterol became exchangeable. In summary, although caveolae/raft lipids were less fluid than those of plasma membranes, sterol domains in caveolae/rafts were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes. Thus caveolae/raft domains isolated without the use of detergents display unique structure, cholesterol domain kinetics and responsiveness to SCP-2 as compared with the parent plasma membrane.

Topics: Animals; Carbocyanines; Carrier Proteins; Caveolae; Cell Line; Cell Membrane; Cholesterol; Diphenylhexatriene; Ergosterol; Fatty Acids, Unsaturated; Fibroblasts; Fluorescence Polarization; Fluorescent Dyes; Gels; Lipids; Membrane Microdomains; Methylamines; Mice; Molecular Probe Techniques; Molecular Probes; Peptides; Propionates; Protein Structure, Tertiary; Solutions; Sterols; Subcellular Fractions

The effect of dye localization and dye distribution on the antineoplastic behavior of photosensitizers was investigated with a homologous series of trimethine thiacarbocyanine dyes in L1210 leukemia and A549 lung carcinoma cells. These dyes were synthesized with N-alkyl groups of different sizes (ethyl to octadecyl) to vary their lipophilic properties without compromising their photophysics. While dyes with smaller N-alkyl groups (ethyl to decyl) were already cytotoxic in the dark, longer chain cyanines exhibited antineoplastic activity only after exposure to light. Results from this study indicate that the switch from dark cytotoxicity to phototoxicity occurred when dyes, due to a decrease in cationic character with increasing size of alkyl substituents, were no longer able to cross the plasma membrane. Dark cytotoxicity decreased with increasing size of N-alkyl groups and was cell-line independent. On the other hand, photodynamic damage varied by several orders of magnitude depending on the cell line and the length of the alkyl substituents. The most effective photosensitizer was the dioctadecyl dye which achieved a 4- to 5-log reduction of leukemia cells, although it had very modest triplet and singlet oxygen quantum yields of 0.008 and 0.006, respectively. This study also showed that photobiological performance can be improved greatly by optimizing dye binding properties via structural modifications.

Topics: Animals; Antineoplastic Agents; Carbocyanines; Fatty Acids, Unsaturated; Fluorescent Dyes; Humans; Leukemia L1210; Lung Neoplasms; Mice; Photochemistry; Structure-Activity Relationship; Tumor Cells, Cultured