A novel methodology for the investigation of intracellular proteolytic processing in intact cells

Taking advantage of the unique spectral properties of the fluorescent probe FL-Bodipy, we have developed a new methodology to study processing of exogenous proteins in intact cells. FL-Bodipy was conjugated to bovine serum albumin (BSA) at a molar ratio of 29 probe molecules to 1 albumin equivalent....

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Bibliographic Details
Published in:European journal of cell biology 1998-02, Vol.75 (2), p.192-197
Main Authors: Reis, Renata C.M., Sorgine, Marcos H.F., Coelho-Sampaio, Tatiana
Format: Article
Language:English
Subjects:
Animals
Boron Compounds - metabolism
Boron Compounds - pharmacokinetics
Cattle
Endopeptidases - metabolism
fluorescence dequenching
Fluorescent Dyes - metabolism
Fluorescent Dyes - pharmacokinetics
Intracellular Fluid
Intracellular proteolysis
macrophages
Macrophages - metabolism
Microscopy, Fluorescence
Protein Processing, Post-Translational
Serum Albumin, Bovine - metabolism
Serum Albumin, Bovine - pharmacokinetics
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Summary:Taking advantage of the unique spectral properties of the fluorescent probe FL-Bodipy, we have developed a new methodology to study processing of exogenous proteins in intact cells. FL-Bodipy was conjugated to bovine serum albumin (BSA) at a molar ratio of 29 probe molecules to 1 albumin equivalent. The resulting conjugate was 98% self-quenched due to fluorescence resonance energy transfer (homotransfer) between neighboring Bodipy molecules. In vitro proteolytic cleavage of the conjugate led to relaxation of self-quenching and to a significant increase in fluorescence. Ftow cytometry and fluorescence microscopy indicated that Bodipy-labeled BSA was readily internalized by J774 macrophages and accumulated in intraceUular compartments. The kinetics of intraceUular degradation of Bodipy-BSA was linear for up to 2 hours and was completely inhibited by a combination of protease inhibitors. Future applications of the methodology reported here may comprise studies of antigen processing and presentation, as weD as the investigation of ceUular events related to processing and disassembly of intraceUular pathogens such as parasites, bacteria and viruses.
ISSN:0171-9335
1618-1298
DOI:10.1016/S0171-9335(98)80061-7