metallothionein and cyanine-dye-3

Herein, we report a new simple and easy-to-use approach for the characterization of protein oligomerization based on fluorescence resonance energy transfer (FRET) and capillary electrophoresis with LED-induced detection. The FRET pair consisted of quantum dots (QDs) used as an emission tunable donor (emission wavelength of 450 nm) and a cyanine dye (Cy3), providing optimal optical properties as an acceptor. Nonoxidative dimerization of mammalian metallothionein (MT) was investigated using the donor and acceptor covalently conjugated to MT. The main functions of MTs within an organism include the transport and storage of essential metal ions and detoxification of toxic ions. Upon storage under aerobic conditions, MTs form dimers (as well as higher oligomers), which may play an essential role as mediators in oxidoreduction signaling pathways. Due to metal bridging by Cd

Topics: Acetates; Animals; Cadmium; Carbocyanines; Dimerization; Electrophoresis, Capillary; Fluorescence Resonance Energy Transfer; Metallothionein; Models, Molecular; Protein Conformation; Quantum Dots; Rabbits; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Static Electricity

Metallothioneins (MTs) are low molecular weight, stress-activated proteins that protect cells against heavy metals, oxidants, and some electrophilic drugs. Both nuclear and cytoplasmic MT phenotypes have been observed in cells even though MTs (6 kDa) are well below the size exclusion limit for diffusion through the nuclear envelope. To study the factors controlling MT subcellular partitioning, we covalently linked MTII to a fluorescent label and examined its subcellular distribution in response both to pharmacologic and physical perturbations. Fluorescent MTII localized to the nucleus of digitonin-permeabilized human SCC25 carcinoma cells, consistent with its endogenous distribution in these cells. Nuclear sequestration of the fluorescent MTII was inhibited by a 100-fold molar excess of unlabeled MTII and by wheat germ agglutinin, indicating a saturable binding mechanism and the involvement of one or more glycoproteins, respectively. Depletion of adenosine triphosphate (ATP) inhibited MTII nuclear localization, implying energy-dependent nuclear translocation or retention of MT. Neither chilling nor the absence of cytosolic extracts inhibited nuclear sequestration of MTII, supporting diffusion-based entry mechanism. In situ biochemical extractions of the nuclear MTII revealed at least two distinct binding activities. Collectively, these data indicate that MTII diffuses into the nucleus of SCC25 cells, where it is selectively and actively retained by nuclear binding factors, imparting its localization phenotype.

Topics: Adenosine Triphosphate; Animals; Biological Transport; Cadmium; Calmodulin; Carbocyanines; Cell Extracts; Cell Line; Cell Membrane Permeability; Cell Nucleus; Cold Temperature; Cytosol; Diffusion; Energy Metabolism; Fluorescent Dyes; Glycosylation; Humans; Metallothionein; Mice; Protein Binding; Wheat Germ Agglutinins