Serum Albumin Binding of Natural Substances and Its Influence on the Biological Activity of Endogenous and Synthetic Ligands for G-Protein-Coupled Receptors
Catherina Caballero-George in Natural Products and Cardiovascular Health, 2018
Nevertheless, it is not known whether BSA modulates the ability of angiotensin II to activate AT1R. In order to systematically investigate this, we have measured angiotensin II-mediated calcium responses in the absence and presence of 0.1% BSA. As read-out system, we used CHO-K1 cells that are stably transfected with the bioluminescent protein aequorin and that upon calcium binding emit a strong light signal that was measured in a 96-well reader (denoted as CHO-AEQ cells). Transfecting these cells with the human AT1R enabled us to measure characteristic transient calcium responses by angiotensin II. The response was determined by calculating the area under the curve of transient rise in luminescence after angiotensin II exposure (Bahem et al., 2015). It appeared that the angiotensin II concentration-response curves were slightly leftward shifted in the presence of 0.1% BSA (Figure 9.1). The corresponding EC50 values were 0.31 and 4.54 nM, respectively, in the presence or absence of BSA, indicating that angiotensin II becomes more potent in the presence of BSA. Although this observation indicates that BSA is indeed capable of binding angiotensin II, the underlying mechanism needs to be further elucidated.
Hypoxia, Free Radicals, and Reperfusion Injury Following Cold Storage and Reperfusion of Livers for Transplantation
John J. Lemasters, Constance Oliver in Cell Biology of Trauma, 2020
In hepatocytes loaded with aequorin, a Ca2+-indicating photoprotein, a biphasic response of free Ca2+ to anoxia in isolated hepatocytes was reported.64 Ca2+ increased initially and then returned toward baseline. Subsequently, as lactate dehydrogenase was released, free Ca2+ increased again. In these experiments, hepatocytes were centrifuged in Ca2+-free, aequorin-containing medium to “gravity load” aequorin. In this work, cytosolic lactate dehydrogenase, but not aequorin, was reportedly released from hepatocytes during anoxic injury. If this is true, aequorin cannot be loaded exclusively into the cytosol by gravity. Therefore, all or part of the Ca2+ changes measured by aequorin may result from changes of Ca2+ in endosomes or other subcellular compartments that aequorin enters while hepatocytes were exposed to high gravitational fields.
Kinetic Thinking: Back to the Future
Clive R. Bagshaw in Biomolecular Kinetics, 2017
Wild-type green fluorescent protein (GFP) provides a good example of excited-state proton transfer, where the dominant (80%) ground-state protonated form, GFPH, absorbs light at 398 nm, but on excitation, it rapidly loses a proton to give the anionic form of the chromophore, GFP−*, that emits at 508 nm [303,703]. While this situation is beneficial to the jelly fish, Aequorea victoria, where it acts as a Förster resonance energy transfer (FRET) acceptor for the chemiluminescent protein, aequorin, excited-state proton transfer makes GFP less convenient as a biotechnological tool. Enhanced GFP (eGFP) was developed by mutagenesis to shift the protonation equilibrium so that the eGFP− form dominated the ground state and the emission at 508 nm was greatly increased when excited at directly 480 nm [704]. Wild-type GFP continues to attract the attention of biophysicists because, on excitation, the released proton is rapidly transferred through a “proton wire” of ionizable side chains and water molecules within the protein core to the surface by a Grotthuss-like mechanism [524,705]. Consequently, GFP acts as a model light-driven proton pump.
Advances in luminescence-based technologies for drug discovery
Published in Expert Opinion on Drug Discovery, 2023
Bolormaa Baljinnyam, Michael Ronzetti, Anton Simeonov
Bioluminescence resonance energy transfer (BRET) is a process where energy transfer occurs from a bioluminescent donor to a fluorescent acceptor. Interestingly, the green fluorescent protein (GFP), isolated by Shimomura and colleagues in early 1960s from the Aequorea jellyfish, emits green light in those jellyfishes by absorbing the excited state energy of the luciferase aequorin, which itself catalyzes the oxidation of the substrate molecule coelenterazine triggering the chemiluminescence process [54].
Modulation of neuromuscular synapses and contraction in Drosophila 3rd instar larvae
Published in Journal of Neurogenetics, 2018
Kiel G. Ormerod, JaeHwan Jung, A. Joffre Mercier
Hewes, Snowdeal, Saitoe, and Taghert (1998) performed a detailed analysis of effects of the peptides encoded in dFMRFa on nerve-evoked contractions of larval body wall muscles. SAPQDFVRSamide had no effect, but the other seven peptides increased contraction amplitude, with thresholds of ∼10 nM for DPKQDFMRFamide, SPKQDFMRFamide, SDNFMRFamide, PDNFMRFamide and SVQDNFMHRamide, and 10-fold lower for TPAEDFMRFamide and MDSNFIRFamide. EC50 values were ∼40 nM for most of the peptides, and their effects were equivalent whether applied separately or in combination, suggesting that the peptides are functionally redundant (Hewes et al., 1998). This view was supported by subsequent work identifying one GPCR that, when expressed in CHO cells and assayed for bioluminescence of co-transfected aequorin, fails to respond to SAPQDFVRSamide but responds to the other peptides encoded in dFMRFa with similar EC50 values, ranging as low as 0.9–2 nM (Cazzamali & Grimmelikhuijzen, 2002; Meeusen et al., 2002). This GPCR also responded to Drosophila myosuppressin, sulfakinin-1 and short neuropeptide F, but at concentrations too high to be considered physiologically relevant (EC50 values 38–110 nM), so it was designated the Drosophila FMRFamide receptor (FR). Genes for two receptors for Drosophila myosuppressin (DmsR-1 and DmsR-2) were subsequently identified, and when expressed in CHO cells and examined with a bioluminescence assay, they failed to respond to FMRFamide, Drosophila short neuropeptide F-1 and perisulfakinin (Egerod et al., 2003a). DmsR-2 was also expressed in HEK cells and assayed for translocation of [beta]-arrestin2-green fluorescent protein ([beta]ARR2-GFP), a protein involved in desensitization of nearly all GPCRs (Kohout & Lefkowitz, 2003). These cells responded to both Drosophila myosuppressin and DPKQDFMRFamide if a G-protein coupled receptor kinase was co-expressed to accelerate [beta]ARR2-GFP translocation (Johnson, Bohn, et al., 2003). HEK cells expressing either DmsR-1 or DmsR-2 also showed decreased cAMP levels in response to both myosuppressin and DPKQDFMRFamide but were ∼10-fold less sensitive to the latter peptide. Thus, many people have favoured the view that there is only one receptor for the peptides encoded in dFMRFa, at least at physiologically relevant peptide concentrations. Receptor and ligand modeling indicates that the five Drosophila peptides containing the sequence ‘FMRFamide’ exhibit subtle differences in docking and linking with the FMRFamide receptor, but they all elicit very similar responses in cardiac bioassays (Maynard et al., 2013).
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