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en: Efficient conversion to Cypridina luciferin from Cypridina luciferyl sulfate, coupled with enzymatic sulfation of acetic acid.

en: In Cypridina (Vargula) hilgendorfii, Cypridina luciferin is converted from Cypridina luciferyl sulfate by a sulfotransferase with adenosine 3', 5'-diphosphate (PAP), and is used for the luminescence reaction of Cypridina luciferase. We found that the luminescence activity of crude extracts of C. hilgendorfii was significantly stimulated by the addition of acetic acid. This stimulation may be explained by an efficient supply of PAP from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) catalyzed by a sulfotransferase. Thus, acetic acid acts as a sulfate acceptor from PAPS, followed by forming acetyl sulfate and PAP. The structure of acetyl sulfate was identified using mass spectrometry and it spontaneously decomposed to acetic acid and free sulfate ion in aqueous solutions. This enzymatic conversion from Cypridina luciferyl sulfate to Cypridina luciferin could be coupled with acetic acid and PAPS by a sulfotransferase.

en: Biochemical and Biophysical Research Communications


en: A novel yellow fluorescent protein of recombinant apoPholasin with dehydrocoelenterazine

en: Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λ = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-HO system, but not in sodium hypochlorite.

ja: Biochemical and Biophysical Research Communications


en: Identification of 3-enol sulfate of Cypridina luciferin, Cypridina luciferyl sulfate, in the sea-firefly Cypridina (Vargula) hilgendorfii

en: Tetrahedron

ja: Tetrahedron


en: Development of simple firefly luciferin analogs emitting blue, green, red, and near-infrared biological window light


en: Identification of cytotoxic dimers in oxidation product from sesamol, a potent antioxidant of sesame oil

en: Journal of Agricultural and Food Chemistry


en: Distinction of Sialyl Anomers on ESI- and FAB-MS/MS: Stereo-Specific Fragmentations

en: Journal of the American Society for Mass Spectrometry


en: Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin

en: The chirality of the luciferin substrate is critical to the luciferin-luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D-luciferin is still unclear, although it can be synthesized in vitro from D-cysteine. Here, we show that the firefly produces both D- and L-luciferin, and that the amount of active D-luciferin increases gradually with maturation stage. Studies of firefly body extracts indicate the possible conversion of L-cysteine via L-luciferin into D-luciferin, suggesting that the biosynthesis is enzymatically regulated by stereoisomeric bio-inversion of L-luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought.

en: FEBS Letters

ja: FEBS Letters


en: Construction of a new firefly bioluminescence system using L-luciferin as substrate

en: Tetrahedron Letters

ja: Tetrahedron Letters


en: Novel relationship beween the antifungal activity and cytotoxicity of marine-derived methabolite xestoquinone and its family

en: Xestoquinone and related metabolites (the xestoquinone family) occur in marine sponges and are known to show a variety of biological activities. In this study, the first comprehensive evaluation of antifungal activity was performed for xestoquinone and nine natural and unnatural analogues in comparison with their cytotoxicity. The cytotoxicity against two human squamous cell carcinoma cell lines, A431 and Nakata, indicated that the terminal quinone structure of the polycyclic molecules was important (xestoquinone, etc.) and that the presence of a ketone group at C-3 of the opposite terminus dramatically diminished the activity (halenaquinone, etc.). In contrast, a ketone group at C-3 enhanced the antifungal activity against the plant pathogen, Phytophthora capsici, regardless of the presence of a quinone moiety. The cytotoxicity and antifungal activity of the xestoquinone family were negatively correlated with each other.

en: Bioscience, Biotechnology, and Biochemistry


en: Firefly luciferase exhibits bimodal action depending on the luciferin chirality

en: Firefly luciferase is able to convert L-luciferin into luciferyl-CoA even under ordinary aerobic luciferin-luciferase reaction conditions. The luciferase is able to recognize strictly the chirality of the luciferin structure, serving as the acyl-CoA synthetase for L-luciferin, whereas d-luciferin is used for the bioluminescence reaction. D-Luciferin inhibits the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin, meaning that both enzyme activities are prevented by the enantiomer of its own substrate.

en: Biochemical and Biophysical Research Communications


en: Bioluminescence activity of Latia luciferin analogues: Replacement of the 2,6,6-trimethylcyclohexene ring onto the methyl-substituted phenyl groups


en: Effects of modification at the fifth residue of mu-conotoxin GIIIA with bulky tags on the electrically stimulated contraction of the rat diaphragm

en: Mu-conotoxin GIIIA, a peptide toxin from the cone snail, blocks muscle-type sodium channels. Thr-5 of mu-conotoxin GIIIA, located on the opposite side of the active site in the globular molecule, was replaced by Cys to which the bulky tags were attached. The tagged mu-conotoxin GIIIA derivatives, except for the phospholipid-tagged one, exerted the biological activity with a potency slightly weaker than natural mu-conotoxin GIIIA. When the biotinylated tags of various lengths were added, the presence of avidin suppressed the action of the biotinylated toxins of <4 nm, but not with 5 nm. The bulky biotinylated tags are useful as a caliper to measure the depth of receptor sites in the channels.

en: The Journal of Peptide Research

ja: The Journal of Peptide Research


en: Synthesis of Latia luciferin benzoate analogues and their bioluminescent activity


en: Synthesis of Biotinylated Xestoquinone That Retains Inhibitory Activity Against Ca2+ ATPase of Skeletal Muscle Myosin

en: Xestoquinone isolated from a marine sponge binds to skeletal muscle myosin and inhibits its Ca(2+) ATPase activity. In this study, we first examined xestoquinone and its analogues to assess the relationships between structure and myosin Ca(2+) ATPase inhibitory activity. On the basis of the resultant data, we then designed a biotinylated xestoquinone analogue. Xestoquinone and its analogues were derived from extracts of the marine sponge Xestospongia sapra. Four xestoquinone analogues with a quinone structure significantly inhibited Ca(2+) ATPase activity. In contrast, four xestoquinone analogues in which the quinone structure was converted to a quinol dimethyl ether did not inhibit Ca(2+) ATPase activity. This suggests that the quinone moiety is essential for inhibitory activity. Then, we synthesized a biotinylated xestoquinone in which a biotin tag was introduced to a site far from the quinone moiety, and this molecule exhibited stronger inhibitory activity than that of xestoquinone. This biotinylated xestoquinone could be useful as a probe in studies of the xestoquinone-myosin binding mode.

en: Bioorganic & Medicinal Chemistry

ja: Bioorganic & Medicinal Chemistry


en: Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization

en: The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.

en: Nature Cell Biology

ja: Nature Cell Biology


en: Identification of biotinylated lysine residues in the photoprotein aequorin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mapping after lysine-specific endopeptidase digestion

en: 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.

en: Analytical Biochemistry: Methods in the Biological Sciences


en: Thiolation of protein-bound carcinogenic aldehyde: An electrophilic acrolein-lysine adduct that covalently binds to thiols

en: Acrolein, a representative carcinogenic aldehyde that could be ubiquitously generated in biological systems under oxidative stress, shows facile reactivity with the epsilon-amino group of lysine to form N(epsilon)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product (Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) J. Biol. Chem. 273, 16058-16066). In the present study, we determined the electrophilic potential of FDP-lysine and established a novel mechanism of protein thiolation in which the FDP-lysine generated in the acrolein-modified protein reacts with sulfhydryl groups to form thioether adducts. When a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, was incubated with acrolein-modified bovine serum albumin in sodium phosphate buffer (pH 7.2) at 37 degrees C, a significant loss of sulfhydryl groups, which was accompanied by the loss of enzyme activity and the formation of high molecular mass protein species (>200 kDa), was observed. The FDP-lysine adduct generated in the acrolein-modified protein was suggested to represent a thiol-reactive electrophile based on the following observations. (i) N(alpha)-acetyl-FDP-lysine, prepared from the reaction of N(alpha)-acetyl lysine with acrolein, was covalently bound to glyceraldehyde-3-phosphate dehydrogenase. (ii) The FDP-lysine derivative reacted with glutathione to form a GSH conjugate. (iii) The acrolein-modified bovine serum albumin significantly reacted with GSH to form a glutathiolated protein. Furthermore, the observation that the glutathiolated acrolein-modified protein showed decreased immunoreactivity with an anti-FDP-lysine monoclonal antibody suggested that the FDP-lysine residues in the acrolein-modified protein served as the binding site of GSH. These data suggest that thiolation of the protein-bound acrolein may be involved in redox alteration under oxidative stress, whereby oxidative stress generates the increased production of acrolein and its protein adducts that further potentiate oxidative stress via the depletion of GSH in the cells.

en: The Journal of Biological Chemistry

ja: The Journal of Biological Chemistry


en: Generation of Polyclonal Antibody against mu-Conotoxin GIIIA Using an Immunogen of [Cys5]mu-Conotoxin GIIIA Site-Specifically Conjugated with Bovine Serum Albumin

en: mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins.

en: Biochemical and Biophysical Research Communications


en: Modification of Arg-13 of mu-conotoxin GIIIA with piperidinyl-Arg analogs and the relation to the inhibition of sodium channels

en: mu-Conotoxin GIIIA, a peptide toxin isolated from the marine snail Conus geographus, preferentially blocks skeletal muscle sodium channels in vertebrates. In this study, analogs of mu-conotoxin GIIIA in which essential Arg-13 was replaced with arginine analogs consisting of a piperidyl framework to regulate length and direction of the side chain were synthesized. Synthesized analogs exhibited similar CD and NMR spectra to that of GIIIA, suggesting a three-dimensional structure identical to that of the native toxin. The biological activities of piperidyl analogs were decreased or lost despite the small change in the side chain of Arg-13. The investigated structure-activity relationships in inhibiting electrically stimulated muscle contraction suggest that the guanidinium group at amino acid position 13 interacts best when spaced with three to four carbons and placed in a vertical direction from the peptide loop. Thus, the position of the guanidinium group at Arg-13 of GIIIA must be located in a certain range for its strong interaction with the channel protein.

en: FEBS Letters


en: Synthesis of [Cys5]mu-conotoxin GIIIA and its derivatives as a probe of Na+ channel analysis

en: The residue of Thr-5 in mu-conotoxin GIIIA (GIIIA), a receptor site I sodium channel blocker, was replaced with Cys. The synthesized [Cys(5)]GIIIA had a similar 3D structure to the native GIIIA, revealed by CD and NMR. [Cys(5)]GIIIA and its tagged peptides inhibited the electrically stimulated contraction of the rat diaphragm with relatively comparable potency to that of GIIIA. Since the contractile response to electrical stimuli is caused by the activation of sodium channels, [Cys(5)]GIIIA could be a prototype for synthesizing useful tools for the analysis of sodium channels. Thus, [Cys(5)]GIIIA could be a prototype for synthesizing useful tools for the analysis of sodium channels.