PubMed · 11101885
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T Smith. 2000. Going green.. https://doi.org/10.1038/81925
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A method for identifying modified lysine residues in a protein, using lysine-specific endopeptidase treatment followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mapping, is described. As a model protein, the photoprotein aequorin was chosen and the N-hydroxysuccinimide ester of biotin was employed to chemically modify the lysine residues. After digestion with lysine-specific endopeptidase, the biotinylated residues of an amino terminus and five potential lysine residues were identified by MALDI-TOF-MS without any other separation procedure.
Fatty acids, which are essential nutritional components, are also involved in cardiovascular and metabolic diseases. Here we report a human cell surface receptor that we name free fatty acid receptor (FFAR), because it is specifically activated by medium to long-chain free fatty acids. The receptor belongs to the class of seven-transmembrane, G-protein coupled receptors (GPCRs) and also mediates responses to antidiabetic drugs of the thiazolidinedione type. It is expressed in skeletal muscle, heart, liver, and pancreatic beta-cells. Stimulation of FFAR increases the intracellular calcium concentration in cells expressing the receptor in a native (pancreatic beta-cell line) or in a recombinant form. In view of the nature of the activating substances, their physiological role in the body, and the tissue distribution of FFAR we suggest the term "nutrient sensing receptor" for receptors acting at the interface between dietary components and signalling molecules.
Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.