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MN2+-ACTIVATED LUMINESCENCE OF THE PHOTOPROTEIN OBELIN [Text] / E. S. VYSOTSKI [et al.] // Arch. Biochem. Biophys. - 1995. - Vol. 316, Is. 1. - P92-99, DOI 10.1006/abbi.1995.1014. - Cited References: 38 . - 8. - ISSN 0003-9861

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
CALCIUM-ACTIVATED PHOTOPROTEINS
   CTENOPHORES MNEMIOPSIS SP
   CA-2+-ACTIVATED PHOTOPROTEIN
   MESSENGER-RNA
   BEROE-OVATA
   AEQUORIN
   PURIFICATION
   PROTEIN
   CDNA
   EXTRACTION
Аннотация: The light emission of obelin may be initiated by Mn2+ under alkaline conditions. The luminescence takes place in a pH range from 7 to 12 with a sharp optimum at 11.75. The first-order rate constant for Mn2+-activated luminescence decay is more than 9 s(-1), while that for Ca2+-activated luminescence decay is only 6.9 s(-1). The Mn2+ concentration-effect curve for obelin determined with simple dilutions of manganese salt is a sigmoid curve, The slope of the curve is moderately dependent on the pH and was not more than 1 within the pH range tested. The maximal light emission, which is initiated by 3.6 X 10(-5) M Mn2+ at pH 11.75 was about 10% of the maximal Ca2+-activated luminescence. Mg2+ ions inhibit the Mn2+-activated luminescence of obelin. The addition of OH. and O-2(-) scavengers did not influence the Mn2+-activated luminescence, but when singlet oxygen quenchers were added, the Mn2+-dependent light emission was inhibited. This suggests that the O-1(2) might be formed and itself be responsible for chromophore oxidation attended with light emission. NEM and Na2S2O4 inhibit the Mn2+-initiated light emission of obelin completely, showing that endogenous hydroperoxide and SH-group(s) of the photoprotein are essential for both Ca2+-activated and Mn2+-activated light emission of obelin. (C) 1995 Academic Press, Inc.
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
VYSOTSKI, E.S.; TROFIMOV, C.P.; BONDAR, V.S.; FRANK, L.A.; MARKOVA, S.V.; ILLARIONOV, B.A.

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Highly active BRET-reporter based on yellow mutant of Renilla muelleri luciferase / E. V. Eremeeva, S. V. Markova, E. S. Vysotski // Dokl. Biochem. Biophys. - 2013. - Vol. 450, Is. 1. - P147-150, DOI 10.1134/S1607672913030095. - Cited References: 14. - This work was supported by the Ministry of Education and Science of the Russian Federation (Government Contract no. 16.512.11.2141) and Council of the President of the Russian Federation on Grants and State Support of Leading Scientific Schools (project no. NSh-64987.2010.4). . - ISSN 1607-6729

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
GREEN-FLUORESCENT PROTEIN
   GENE-EXPRESSION
   CDNA
   CLONING
   BIOLUMINESCENCE
   RENIFORMIS

Scopus
Держатели документа:
[Eremeeva, E. V.
Markova, S. V.
Vysotski, E. S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
[Eremeeva, E. V.
Markova, S. V.
Vysotski, E. S.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
ИБФ СО РАН
Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036, Russian Federation
Siberian Federal University, Svobodnyi pr. 79, Krasnoyarsk, 660041, Russian Federation : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Markova, S.V.; Vysotski, E.S.

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High-active truncated luciferase of copepod Metridia longa / S. V. Markova, L. P. Burakova, E. S. Vysotski // Biochem. Biophys. Res. Commun. - 2012. - Vol. 417, Is. 1. - P98-103, DOI 10.1016/j.bbrc.2011.11.063. - Cited References: 31. - This study was supported by the Grants 16.512.11.2141 and 64987.2010.4 of the Ministry of Education and Science of Russian Federation. . - ISSN 0006-291X

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
COELENTERAZINE-BINDING PROTEIN
   REPORTER-GENE-EXPRESSION
   RENILLA LUCIFERASE
   GAUSSIA LUCIFERASE
   LIGHT-EMITTER
   IN-VIVO
   BIOLUMINESCENCE
   PHOTOPROTEINS
   CDNA
   SUBSTRATE
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Mammalian expression -- Secretion
Аннотация: The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coil cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme. (C) 2011 Elsevier Inc. All rights reserved.

Держатели документа:
[Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
Siberian Fed Univ, Dept Biophys, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Burakova, L.P.; Vysotski, E.S.

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Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase [Text] / G. A. Stepanyuk [et al.] // Anal. Bioanal. Chem. - 2010. - Vol. 398, Is. 4. - P1809-1817, DOI 10.1007/s00216-010-4106-9. - Cited References: 39. - This work was supported by grant 09-04-12022 of the Russian Foundation for Basic Research, "Molecular and Cell Biology" program of Russian Academy of Sciences, by the SB RAS grant No. 2, and by the SB RAS Lavrentiev grant for Young Scientists. . - ISSN 1618-2642

РУБ Biochemical Research Methods + Chemistry, Analytical
Рубрики:
GREEN-FLUORESCENT PROTEIN
   BIOLUMINESCENT REPORTER
   CA2+-REGULATED PHOTOPROTEINS
   RENIFORMIS LUCIFERASE
   RECOMBINANT OBELIN
   GENE-EXPRESSION
   IN-VIVO
   CDNA
   CLONING
   PURIFICATION
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Calcium -- Imaging
Аннотация: It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca2+-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 degrees C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

Держатели документа:
[Stepanyuk, Galina A.
Malikova, Natalia P.
Markova, Svetlana V.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Photobiol Lab, Krasnoyarsk 660036, Russia
[Unch, James] Promega Biosci LLC, San Luis Obispo, CA 93401 USA
[Lee, John] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Unch, J...; Malikova, N.P.; Markova, S.V.; Lee, J...; Vysotski, E.S.

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Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay [Text] / V. V. Borisova [et al.] // Photochem. Photobiol. Sci. - 2008. - Vol. 7, Is. 9. - P1025-1031, DOI 10.1039/b807271j. - Cited References: 19. - The work was supported by Bayer AG, by the Russian Foundation for Basic Research grants 05-04-48271 and 06-04-08076, by the joint grant 06-04-89502 of the Russian Foundation for Basic Research and Taiwan National Science Council, and by the "Molecular and Cellular Biology" program of the Russian Academy of Sciences. . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
Рубрики:
BIOLUMINESCENT REPORTER
   GAUSSIA LUCIFERASE
   CDNA
   PROTEINS
   CLONING
   OVEREXPRESSION
   PURIFICATION
   MUTAGENESIS
   ENZYME
   OBELIN
Аннотация: The recombinant coelenterazine-dependent luciferases (isoforms MLuc 164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc09) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca2+-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a "substrate" provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35-40% of the initial activity The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.

Держатели документа:
[Borisova, Vasillisa V.
Frank, Ludmila A.
Markova, Svetlana V.
Burakova, Ludmilla P.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk 660036, Russia
[Frank, Ludmila A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Borisova, V.V.; Frank, L.A.; Markova, S.V.; Burakova, L.P.; Vysotski, E.S.

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Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay [Text] / L. A. Frank [et al.] // Anal. Bioanal. Chem. - 2008. - Vol. 391, Is. 8. - P2891-2896, DOI 10.1007/s00216-008-2223-5. - Cited References: 22 . - ISSN 1618-2642

РУБ Biochemical Research Methods + Chemistry, Analytical
Рубрики:
ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   CRYSTAL-STRUCTURE
   BIOLUMINESCENCE
   AEQUORIN
   IMMUNOASSAY
   EXPRESSION
   CDNA
   PURIFICATION
   CLONING
Кл.слова (ненормированные):
Ca(2+)-regulated photoprotein -- bioluminescence -- dual-color assay
Аннотация: Two kinds of Ca(2+)-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (lambda(max)=390 nm) and the mutant Y139F emits greenish light (lambda (max)=498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones-follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca(2+) solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay.

Держатели документа:
[Frank, Ludmila A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
[Frank, Ludmila A.
Borisova, Vasilisa V.
Markova, Svetlana V.
Malikova, Natalia P.
Stepanyuk, Galina A.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Borisova, V.V.; Markova, S.V.; Malikova, N.P.; Stepanyuk, G.A.; Vysotski, E.S.

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All three Ca2+-binding loops of photoproteins bind calcium ions: The crystal structures of calcium-loaded apo-aequorin and apo-obelin [Text] / L. . Deng [et al.] // Protein Sci. - 2005. - Vol. 14, Is. 3. - P663-675, DOI 10.1110/ps.041142905. - Cited References: 46 . - ISSN 0961-8368

РУБ Biochemistry & Molecular Biology
Рубрики:
RAY CRYSTALLOGRAPHIC ANALYSIS
   ANGSTROM RESOLUTION
   SEQUENCE-ANALYSIS
   CA2+-REGULATED PHOTOPROTEINS
   CA2+-DISCHARGED PHOTOPROTEIN
   LUMINESCENT PROTEIN
   MODULATED PROTEINS
   ELECTRON-DENSITY
   CLONING
   CDNA
Кл.слова (ненормированные):
bioluminescence -- EF-hand -- fluorescent protein -- proton relay -- calcium-binding loops -- aequorin -- obelin -- diffraction
Аннотация: The crystal structures of calcium-loaded apo-aequorin and apo-obelin have been determined at resolutions 1.7 Angstrom and 2.2 Angstrom. respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-proteins retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hydroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with the product, coelenteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these subtle shifts are the basis of the ability of these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Univ Georgia, Dept Chem, Athens, GA 30602 USA
Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Deng, L...; Vysotski, E.S.; Markova, S.V.; Liu, Z.J.; Lee, J...; Rose, J...; Wang, B.C.

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Conjugates of the Ca2+-regulated photoprotein obelin with immunoglobulins: Synthesis and use as labels in bioluminescent immunoassay [Text] / L. A. Frank, A. I. Petunin, E. S. Vysotski // Russ. J. Bioorg. Chem. - 2004. - Vol. 30, Is. 4. - P327-331, DOI 10.1023/B:RUBI.0000037257.80835.7a. - Cited References: 16 . - ISSN 1068-1620

РУБ Biochemistry & Molecular Biology + Chemistry, Organic
Рубрики:
ESCHERICHIA-COLI
   PURIFICATION
   AEQUORIN
   PROTEIN
   CDNA
Кл.слова (ненормированные):
bioluminescent immunoassay -- obelin -- thyroid stimulating hormone
Аннотация: An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 muIU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
DIAS Ltd, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Petunin, A.I.; Vysotski, E.S.

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Preparation and X-ray crystallographic analysis of the Ca2+-discharged photoprotein obelin [Text] / L. . Deng [et al.] // Acta Crystallogr. Sect. D-Biol. Crystallogr. - 2004. - Vol. 60. - P512-514, DOI 10.1107/S090744490302852X. - Cited References: 18 . - ISSN 0907-4449

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Biophysics + Crystallography
Рубрики:
VIOLET BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   W92F OBELIN
   AEQUORIN
   SEQUENCE
   PROTEIN
   CLONING
   CDNA
Аннотация: Ca2+-regulated photoproteins belong to the EF-hand Ca2+-binding protein family. The addition of calcium ions initiates bright blue bioluminescence of the photoproteins, a result of the oxidative breakdown of coelenterazine peroxide to coelenteramide. Crystals of the Ca2+-discharged W92F mutant of obelin from Obelia longissima have been grown, representing the first crystallization of a photoprotein after the Ca2+-triggered bioluminescence. A green fluorescence observed from the crystals clearly demonstrates that coelenteramide, the bioluminescence product of coelenterazine peroxide, is bound within the protein. The diffraction pattern exhibits tetragonal Laue symmetry. Systematic absences indicate that the space group is either P4(3)2(1)2 or P4(1)2(1)2. The unit-cell parameters are a=b=53.4, c=144.0 Angstrom. The crystals diffract to 1.9 Angstrom resolution.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Univ Georgia, Dept Chem, Athens, GA 30602 USA
Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Deng, L...; Markova, S.V.; Vysotski, E.S.; Liu, Z.J.; Lee, J...; Rose, J...; Wang, B.C.

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10.

Bioluminescent immunoassay of thyrotropin and thyroxine using obelin as a label [Text] / L. A. Frank, A. I. Petunin, E. S. Vysotski // Anal. Biochem. - 2004. - Vol. 325, Is. 2. - P240-246, DOI 10.1016/j.ab.2003.11.003. - Cited References: 16 . - ISSN 0003-2697

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Chemistry, Analytical
Рубрики:
AEQUORIN
   PURIFICATION
   PROTEIN
   CDNA
Кл.слова (ненормированные):
obelin -- human thyrotropin -- thyroxine -- immunoassay -- bioluminescence
Аннотация: Solid-phase bioluminescent immunoassay of thyroid hormones, human thyrotropin (hTSH) and two forms of thyroxine (T4), whose determinations are vitally important for diagnostics of thyroid diseases and the efficiency of treatment, is described. The recombinant obelin, a Ca2+-regulated photoprotein originally derived from the luminous marine hydroid Obelia longissima, is employed as a bioluminescent label. To produce obelin conjugates with anti-hTSH, anti-T4 immunoglobulins (IgG), and T4, additional SH groups are introduced into the obelin molecule using Traut's reagent (2-iminothiolane) and then obelin possessing extra SH groups is conjugated with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate-activated IgGs or T4. The total yield of obelin conjugates determined by luminescent activity is 60-65% after all chemical and purification procedures. The obtained conjugates are stable to lyophilization and in solution for at least 9 months at 4 degreesC, with loss of activity not exceeding 10%. The application of obelin conjugates for determination of the hTSH, total T4, and free T4 in standard, control, and patient sera displays high sensitivity and reproducibility of results. The results of bioluminescent immunoassays are closely comparable to those obtained by the radioimmunoassay method (R = 0.95-0.99). (C) 2003 Elsevier Inc. All rights reserved.

Держатели документа:
Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
DIAS Ltd, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Petunin, A.I.; Vysotski, E.S.

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11.

Obelin from the bioluminescent marine hydroid Obelia geniculata: Cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins [Text] / S. V. Markova [et al.] // Biochemistry. - 2002. - Vol. 41, Is. 7. - P2227-2236, DOI 10.1021/bi0117910. - Cited References: 54 . - ISSN 0006-2960

РУБ Biochemistry & Molecular Biology
Рубрики:
AMINO-ACID-SEQUENCE
   CALCIUM-ACTIVATED PHOTOPROTEINS
   CTENOPHORES MNEMIOPSIS SP
   CA-2+-ACTIVATED PHOTOPROTEIN
   LUMINESCENT PROTEIN
   BINDING-PROTEIN
   BEROE-OVATA
   AEQUORIN
   CDNA
   PURIFICATION
Аннотация: A cDNA encoding the Ca2+-regulated photoprotein of the bioluminescent marine hydroid Obelia geniculata was cloned and sequenced. The cDNA is a 774 bp fragment containing two overlapping open reading frames, one of which contained 585 bp encoding a 195 amino acid polypeptide which obviously has the primary structure of the apoprotein of a calcium-regulated photoprotein. Many of the residues are identical to those in other Ca2+-regulated photoproteins: 86% compared with that from Obelia longissima, 76% with that from Clytia (Phialidium), 64% with that from Aequorea, and 64% with that from Mitrocoma (Halistaura). The obelin from O. geniculata was overexpressed in Escherichia coli, refolded from inclusion bodies, and purified. The yield of highly purified recombinant protein was 55-80 mg/L of LB medium. O. geniculata obelin has absorption maxima at 280 and 460 nm and a shoulder at approximately 310 nm. The calcium-discharged protein loses visible absorption but exhibits a new absorption maximum at 343 nm. The bioluminescence of the obelin from O. geniculata is blue (lambda(max) = 495 nm). In contrast, the fluorescence of the calcium-discharged protein is yellow-green (lambda(max) = 520 nm; excitation at 340 nm). This is in sharp contrast to aequorin in which the bioluminescence and fluorescence emission spectra of the calcium-discharged protein are almost identical (lambda(max) = 465 nm). The Ca2+ concentration-effect curve for O. geniculata obelin is similar to those of many other photoproteins: at [Ca2+] below approximately 10(-8) M, calcium-independent luminescence is observed, and at [Ca2+] approximately 10(-3) M, the luminescence reaches a maximum. Between these extremes, the curve spans a vertical range of almost 8 log units with a maximum slope on a log-log plot of about 2.5. In the absence of Mg2+ the rate constant for the rise of bioluminescence determined by the stopped-flow technique is about 450 s(-1). The effects of Mg2+ on the kinetics of bioluminescence are complicated, but at all concentrations studied they are relatively small compared to the corresponding effects on aequorin luminescence. At least with respect to speed and sensitivity to Mg2+, the obelins from both O. longissima and O. geniculata would appear to be more suitable than aequorin for use as intracellular Ca2+ indicators.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk 660036, Russia
Univ Washington, Friday Harbor Labs, Friday Harbor, WA 98250 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Vysotski, E.S.; Blinks, J.R.; Burakova, L.P.; Wang, B.C.; Lee, J...

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12.

Preparation and X-ray crystallographic analysis of recombinant obelin crystals diffracting to beyond 1.1 angstrom [Text] / E. S. Vysotski [et al.] // Acta Crystallogr. Sect. D-Biol. Crystallogr. - 2001. - Vol. 57. - P1919-1921, DOI 10.1107/S0907444901016523. - Cited References: 16 . - ISSN 0907-4449

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Biophysics + Crystallography
Рубрики:
PHOTOPROTEIN AEQUORIN
   LONGISSIMA
   EVOLUTION
   CDNA
Аннотация: Crystals of recombinant obelin, the Ca2+-regulated photoprotein from the marine hydroid Obelia longissima, have been grown from a solution containing PEG 8000 and potassium phosphate. Hexamine-cobalt trichloride was used as an additive to increase the chance of crystallization. The crystals grow in a light yellow cubic form (0.5 x 0.5 x 0.45 mm) which diffracts to beyond 1.1 Angstrom resolution. The crystals belong to the space group C2, with unit-cell parameters a=83.43, b=54.92, c=52.99 Angstrom, beta = 112.00 degrees. The asymmetric unit contains one molecule. Crystals exposed to calcium ion before and after X-ray irradiation emit light, confirming that the crystals consist of an active photoprotein.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Vysotski, E.S.; Liu, Z.J.; Rose, J...; Wang, R.C.; Lee, J...

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13.

Cotranslational formation of active photoprotein obelin in a cell-free translation system: Direct ultrahigh sensitive measure of the translation course [Text] / N. G. Berestovskaya [et al.] // Anal. Biochem. - 1999. - Vol. 268, Is. 1. - P72-78, DOI 10.1006/abio.1998.3051. - Cited References: 22 . - ISSN 0003-2697

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Chemistry, Analytical
Рубрики:
SEQUENCE-ANALYSIS
   MESSENGER-RNA
   CA-2+-ACTIVATED PHOTOPROTEIN
   LIGHT-EMISSION
   AEQUORIN
   CDNA
   CLONING
   EXPRESSION
Аннотация: Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes. (C) 1999 Academic Press.

Держатели документа:
Russian Acad Sci, Branch Inst Bioorgan Chem, Pushchino 142292, Russia
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
Tech Univ Berlin, Inst Biochem & Mol Biol, D-10587 Berlin, Germany
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Berestovskaya, N.G.; Shaloiko, L.A.; Gorokhovatsky, A.Y.; Bondar, V.S.; Vysotski, E.S.; Maximov, J.E.; von Doehren, H...; Alakhov, Y.B.

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14.

OBELIN MESSENGER-RNA - A NEW TOOL FOR STUDIES OF TRANSLATION IN CELL-FREE SYSTEMS [Text] / S. V. MATVEEV [et al.] // Anal. Biochem. - 1995. - Vol. 231, Is. 1. - P34-39, DOI 10.1006/abio.1995.1499. - Cited References: 17 . - ISSN 0003-2697

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Chemistry, Analytical
Рубрики:
MESSENGER-RNA
   AEQUORIN
   PROTEIN
   CLONING
   CDNA
Аннотация: Obelin mRNA obtained in vitro with the aid of SP6 RNA polymerase was translated in a wheat germ cell-free system, Only the polypeptide with a molecular mass of about 20 kDa was synthesized. The activation of apoobelin with a synthetic coelenterazine revealed a luminescence activity initiated by calcium. The specific activity was 3.6 +/- 0.4 x 10(15) photons per mg of the in vitro synthesized obelin (k = 6.9 s(-1)). The luminescence of the obelin was in a good correlation with the protein concentration calculated by the incorporation of [C-14]Leu. The determination of the amount of de novo synthesized obelin based on measurement of its luminescence is one-thousand times more sensitive than the approach based on the incorporation of labeled amino acid. Thus, obelin mRNA has some advantages for evaluating the efficiency of cell-free translation when compared with standard methods. (C) 1995 Academic Press, Inc.

Держатели документа:
RUSSIAN ACAD SCI,INST BIOPHYS,KRASNOYARSK 660036,RUSSIA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
MATVEEV, S.V.; ILLARIONOV, B.A.; VYSOTSKI, E.S.; BONDAR, V.S.; MARKOVA, S.V.; ALAKHOV, Y.B.

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15.

Role of conservative residue Cys158 in the formation of an active photoprotein complex of obelin [Text] / V. S. Bondar [et al.] // Biochem.-Moscow. - 2001. - Vol. 66, Is. 9. - P1014-1018, DOI 10.1023/A:1012377827626. - Cited References: 21 . - ISSN 0006-2979

РУБ Biochemistry & Molecular Biology
Рубрики:
CDNA
   EXPRESSION
   AEQUORIN
   SEQUENCE
   CLONING
Кл.слова (ненормированные):
photoproteins -- obelin -- apoobelin mutants -- bioluminescence
Аннотация: Using site directed mutagenesis, the conservative residue Cys158 of recombinant apoobelin was substituted for sera ine (C158S, S-mutant) or alanine (C158A, A-mutant). These point mutations resulted in significant changes in the apoobelin structure accompanied by slowing of photoprotein complex formation, decrease of its stability, and changing of its bioluminescence characteristics. The enzymatic properties of the photoprotein decreased in the series: wild-type protein > S-mutant > A-mutant. This is consistent with rank of nucleophilicity SH > OH > CH3 of cysteine, serine, and alanine side chain functional groups, respectively. Possible mechanisms of the involvement of the apoobelin Cys158 SH-group in the formation of the enzyme-substrate complex are considered.

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Bondar, V.S.; Purtov, K.V.; Malikova, N.P.; Frank, L.A.; Illarionov, B.A.

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16.

Bioluminescent reporters for identification of gene allelic variants / V. V. Krasitskaya [et al.] // Russ. J. Bioorg. Chem. - 2012. - Vol. 38, Is. 3. - P298-305, DOI 10.1134/S1068162012030090. - Cited References: 13. - The authors thank the staff of Hematology Research Center (Krasnoyarsk Branch of Russian Academy of Medical Sciences) for providing DNA samples. The work was supported by the Integration Interdisciplinary Project of Siberian Branch of the Russian Academy of Sciences No. 76 and the Krasno yarsk Regional Fund for the support of scientific and technological activities. . - ISSN 1068-1620

РУБ Biochemistry & Molecular Biology + Chemistry, Organic
Рубрики:
COELENTERAZINE-BINDING PROTEIN
   RENILLA-MUELLERI
   LUCIFERASE
   PURIFICATION
   SUBSTRATE
   CLONING
   CDNA
Кл.слова (ненормированные):
SNP -- PEXT reaction -- obelin -- luciferase -- bioluminescent microassay
Аннотация: A method for single nucleotide polymorphism identification was developed, which was based on the primer extension reaction (PEXT) followed by bioluminescent solid-phase microassay. Recombinant Ca2+-regulated photoprotein obelin and coelenterazine-dependent Renilla muelleri luciferase were used as reporters. The study was performed as an example of SNP genotyping of the human F5 gene encoding human Factor V Leiden polymorphism 1691 G -> A (R506Q). Genomic DNA was amplified by PCR using primers flanking polymorphic site of 140 base pairs. PCR products were used as templates for two PEXT reactions using two primers containing 3'-terminal nucleotides, which were complementary to either normal or mutant alleles. If the template and allele-specific primer were completely complementary, the latter was elongated with DNA polymerase. The resulting extension product contained biotin residue due to the presence of biotinylated deoxyuridine triphosphate (B-dUTP) in the reaction mixture. The products were analyzed using obelin-streptavidin conjugates. The optimal PEXT-reaction conditions were found, which ensured a high reliability of SNP genotyping. A new approach to simultaneously revealing both alleles in one well was developed using two bioluminescent reporters. The efficiency of the proposed approach was shown in the study of clinical DNA samples.

Держатели документа:
[Krasitskaya, V. V.
Burakova, L. P.
Frank, L. A.] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Akademgorodok, Russia
[Pyshnaya, I. A.] Russian Acad Sci, Siberian Branch, Inst Chem Biol & Fundamental Med, Novosibirsk 630090, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Krasitskaya, V.V.; Burakova, L.P.; Pyshnaya, I.A.; Frank, L.A.

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17.

    The C-terminal tyrosine deletion in mitrocomin increases its bioluminescent activity [Text] / L. . Burakova [et al.] // Luminescence. - 2014. - Vol. 29. - P84-84. - Cited References: 6 . - ISSN 1522-7235. - ISSN 1522-7243
Рубрики:
PHOTOPROTEIN
   EXPRESSION
   AEQUORIN
   CLONING
   CDNA

WOS
Держатели документа:
[Burakova, Liudmila
Natashin, Pavel
Markova, Svetlana
Eremeeva, Elena
Vysotsky, Eugene] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk, Russia
[Burakova, Liudmila
Natashin, Pavel
Markova, Svetlana
Eremeeva, Elena
Vysotsky, Eugene] Siberian Fed Univ, Krasnoyarsk, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Burakova, L...; Natashin, P...; Markova, S...; Eremeeva, E...; Vysotsky, E...

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18.

Model of the active site of firefly luciferase [Text] / T. P. Sandalova, N. N. Ugarova // Biochem.-Moscow. - 1999. - Vol. 64, Is. 8. - P. 962-967. - Cited References: 20 . - ISSN 0006-2979

РУБ Biochemistry & Molecular Biology
Рубрики:
ESCHERICHIA-COLI
   SEQUENCE
   CLONING
   ENZYME
   CDNA
   SUPERFAMILY
Кл.слова (ненормированные):
bioluminescence -- firefly luciferase -- ATP -- luciferin -- spatial structure -- active site -- enzyme-substrate complex
Аннотация: A model for the spatial structure of firefly luciferase-ATP-luciferin complex is suggested using the coordinates of unliganded luciferase and the enzyme-substrate complex of the adenylating subunit of gramicidin S synthetase known from the literature. Conformational changes in luciferase can occur during substrate binding resulting in a relative orientation of two luciferase domains similar to that in case of the AMP-phenylalanine-synthetase complex. The model is consistent with data on the physicochemical properties of firefly luciferase and its complexes with the substrates.

WOS
Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
Karolinska Inst, S-17177 Stockholm, Sweden
Moscow MV Lomonosov State Univ, Sch Chem, Moscow 119899, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Sandalova, T.P.; Ugarova, N.N.

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19.

Cotranslational formation of active photoprotein obelin in a cell-free translation system: Direct ultrahigh sensitive measure of the translation course [Text] / N. G. Berestovskaya [et al.] // Anal. Biochem. - 1999. - Vol. 268, Is. 1. - P. 72-78, DOI 10.1006/abio.1998.3051. - Cited References: 22 . - ISSN 0003-2697

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Chemistry, Analytical
Рубрики:
SEQUENCE-ANALYSIS
   MESSENGER-RNA
   CA-2+-ACTIVATED PHOTOPROTEIN
   LIGHT-EMISSION
   AEQUORIN
   CDNA
   CLONING
   EXPRESSION
Аннотация: Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes. (C) 1999 Academic Press.

WOS
Держатели документа:
Russian Acad Sci, Branch Inst Bioorgan Chem, Pushchino 142292, Russia
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
Tech Univ Berlin, Inst Biochem & Mol Biol, D-10587 Berlin, Germany
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Berestovskaya, N.G.; Shaloiko, L.A.; Gorokhovatsky, A.Y.; Bondar, V.S.; Vysotski, E.S.; Maximov, J.E.; von Doehren, H...; Alakhov, Y.B.

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20.

Conjugates of the Ca2+-regulated photoprotein obelin with immunoglobulins: Synthesis and use as labels in bioluminescent immunoassay [Text] / L. A. Frank, A. I. Petunin, E. S. Vysotski // Russ. J. Bioorg. Chem. - 2004. - Vol. 30, Is. 4. - P. 327-331, DOI 10.1023/B:RUBI.0000037257.80835.7a. - Cited References: 16 . - ISSN 1068-1620

РУБ Biochemistry & Molecular Biology + Chemistry, Organic
Рубрики:
ESCHERICHIA-COLI
   PURIFICATION
   AEQUORIN
   PROTEIN
   CDNA
Кл.слова (ненормированные):
bioluminescent immunoassay -- obelin -- thyroid stimulating hormone
Аннотация: An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 muIU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).

WOS
Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
DIAS Ltd, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Petunin, A.I.; Vysotski, E.S.

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