Flavin mononucleotide reductase of luminous bacteria

Duane, Warren C.; Hastings, J. Woodland

doi:10.1007/bf01731866

Cited by 85 publications

(60 citation statements)

References 25 publications

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“…The molecular weights as estimated by linear sucrose-gradient centrifugation using yeast alcohol dehydrogenase as reference were about 1.6 times larger (31 000 and 63000). Our values obtained for the NADH-specific FMN reductase agree quite well with those reported by Puget and Michelson [15] for their NADI-I : flavin oxidoreductase (23500) and Duane and Hastings [3] for their …”

Section: Resultssupporting

confidence: 91%

“…FMNH2, a long-chain aldehyde, oxygen and luciferase. FMNH, which readily oxidises in the presence of O2 is generated by the oxidation of NADH or NADPH through an enzyme which has been given the names of FMN reductase or NAD(P)H dehydrogenase [3]. In wildtype Photobacterium fischeri, it is difficult to separate the dehydrogenase activity from luciferase, suggesting a possible association in vivo between the two activities [4].…”

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confidence: 99%

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Identification of NADH‐Specific and NADPH‐Specific FMN Reductases in Beneckea harveyi

Gerlo¹,

Charlier²

1975

European Journal of Biochemistry

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Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH-specific enzyme is more efficient than the NADPH-specific one with respect to the bioluminescent reaction.Some years ago Strehler and Cormier [l] and McElroy et al. [2] identified the components required for maximum luminescence in a cell-free extract of Photobacterium fischeri, i.e. FMNH2, a long-chain aldehyde, oxygen and luciferase. FMNH, which readily oxidises in the presence of O2 is generated by the oxidation of NADH or NADPH through an enzyme which has been given the names of FMN reductase or NAD(P)H dehydrogenase [3]. In wildtype Photobacterium fischeri, it is difficult to separate the dehydrogenase activity from luciferase, suggesting a possible association in vivo between the two activities [4]. Using a purification procedure adapted from the method of Gunsalus et al. [5], we were able to separate and characterize NADH-specific and NADPH-specific FMN reductase activities in Beneckea harveyi. MATERIALS AND METHODS Growth and Harvesting of CellsBeneckea harveyi cells (previously described as Photobacterium fischeri strain MAV [6] and recently identified by Reichelt and Bauinann [7]), kindly provided by Dr J. W. Hastings, were subcultured in the solid agar medium previously described [5]. The cells were grown at 25 "C with vigorous aeration; anti-foam was added to the medium. Cells were cooled at the time of maximum luminescence, at a density of 1 x lo9 cells/ml, and harvested by continuous Enzymes. Luciferase; NADH-specific and NADPH-specific F M N reductase (EC 1.6.99.-); alcohol dehydrogenase (EC 1.1.1.1).Eur. J. Biochem. 57 (1975) centrifugation in a Sorvall centrifuge (rotor SS 34) at a speed of 16000 rev./min and a flow rate of 250 ml/ min. The yield of packed wet cells was about 4 g/1 medium. Purification ProcedureWe followed the method of Gunsalus-Miguel et al. [5], with minor modifications for the purification of luciferase. Frozen cells were thawed and lysed in cold water adjusted to pH 7.0 with NaOH and containing 0.1 mM dithioerythritol, 5 mM MgSO, and a trace of DNase, in a ratio of 6 ml/g wet pellet. After overnight stirring at 4 " C , EDTA was added to a final concentration of 15 mM. The crude extract was centrifuged for 30 min at 8000 rev./min. Dry DEAE-cellulose (Whatman DE-32) was added to the supernatant (0.22 g/g wet pellet); the pH was maintained at 7.0 by thc addition of 0.5 N acetic acid. Protein was extracted batchwise by increasing phosphate concentrations (0.1, 0.15, 0.5 M potassium phosphate pH 7.0). The fraction extracted by 0.5 M phosphate was then fractionated with ammonium sulfate (special enzyme grade, Mann Research Laboratories). Material precipitating between 40 and 75% saturation was dissolved in 0.25 M phosphate buffer pH 7.0 (0.1 mM dithioerythritol) and dialysed for 16 h against a large volume of the s...

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Section: Resultssupporting

confidence: 91%

mentioning

confidence: 99%

Identification of NADH‐Specific and NADPH‐Specific FMN Reductases in Beneckea harveyi

Gerlo¹,

Charlier²

1975

European Journal of Biochemistry

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“…Subsequent studies indicated that FMN (12) and aldehyde (13) were required for light emission. The NADH-FMN oxidoreductase has recently been partially purified by Duane and Hastings from Beneckea harveyi and Photobacterium fischeri (14). Puget and Michelson have purified the enzyme from Photobacterium sepia (15).…”

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confidence: 99%

“…The range of linearity is 0.1 M to 2 mM. Duane and Hastings reported that this reductase is 10-fold more active with NADH as a substrate (14). It is possible that with the reductase from Photobacterium fischeri which is more active with NADPH, the sensitivity for assaying this compound can be greatly increased (14).…”

mentioning

confidence: 99%

“…Duane and Hastings reported that this reductase is 10-fold more active with NADH as a substrate (14). It is possible that with the reductase from Photobacterium fischeri which is more active with NADPH, the sensitivity for assaying this compound can be greatly increased (14). There may be two different reductases in these bacteria since McElroy and Green observed that partial purification of the enzyme resulted in a relative increased response to NADPH (25).…”

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Immobilization of bacterial luciferase and FMN reductase on glass rods.

Jablonski¹,

DeLuca²

1976

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial luciferase and NAD(P)H: FMN oxidoreductase isolated from Beneckea harveyi were covalently linked via diazotization to arylamine porous glass beads which had been cemented onto plain glass rods. These immobilized enzymes are individually active and also function to produce light via a coupled reaction utilizing NADH or NADPH. These enzymes have properties similar to the soluble forms with regard to pH and substrate optima and also exhibit linearity in peak intensity of the initial flash of light emitted as a function of NADH or NADPH concentration. Linearity with NADH is obtained in the range of 1 pmol to 50 nmol, and between 10 pmol to 200 nmol for NADPH. The bound enzymes are stable and reusable. This immobilized system offers a rapid and inexpensive method of assaying low concentrations of NADH and NADPH, and also serves as a model to study the association of these two enzymes. Bacterial luciferase catalyzes the oxidation of FMNH2 in the presence of a long chain aldehyde with the emission of light (1-4). The aldehyde is converted to the corresponding acid (5-7) and the emitted light has an emission peak at 490 nm. Because of the rapid autoxidation of FMNH2 (8) in an Aminco Chem-Glo photometer and recorded on an Aminco recorder. The initial peak light intensity was linear with respect to added luciferase in the range of 8 ng to 8 ,ug/ml using this instrument. Immobilized luciferase was assayed with the same concentrations of substrates. The rod containing the glass beads was placed in a test tube in the photometer and FMNH2 was injected.Soluble reductase was assayed by measuring the rate of disappearance of absorption at 340 nm in a Cary model 14 recording spectrophotometer. The reaction was initiated by adding 0.1 ml of NADH to 1 ml of 0.015 M phosphate buffer at pH 7.0, containing 70 ,AM EDTA, 0.4 mg of reductase, and FMN. Final concentrations were 0.2 mM NADH, 0.13 mM FMN. When the immobilized enzyme was assayed, the rod containing the enzyme (see below) was dipped into the cuvet which was mixed for 1-min intervals, then removed and the absorbance at 340 measured. This assay gave data that when plotted showed linearity for at least 3 min.The coupled assay was measured by peak light intensity obtained following injection of NADH into 0.5 ml of 0.1 M phosphate buffer at pH 7.0 containing 7.5 Mug of reductase, 5 ,ug luciferase, 2.3 MuM FMN, and 0.0005% decanal. When the immobilized enzyme was being assayed, the rod was immersed in the solution containing FMN and aldehyde, and NADH was injected directly into the solution.Beneckea harveyi strain no. 392 (19) was obtained from K. Nealson of the Scripps Institution of Oceanography. Large quantities of the bacteria were-grown in complete media essentially as described by Farghaly (20) The reductase was separated from the luciferase during chromatography on DEAE-Sephadex, the last step in the purification (22). The peak of reductase activity eluted between 1000 and 1325 ml of column eluant, before the luciferase was eluted. The peak tubes were com...

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2008

Coupled Bioluminescent Assays

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Flavin mononucleotide reductase of luminous bacteria

Cited by 85 publications

References 25 publications

Identification of NADH‐Specific and NADPH‐Specific FMN Reductases in Beneckea harveyi

Identification of NADH‐Specific and NADPH‐Specific FMN Reductases in Beneckea harveyi

Immobilization of bacterial luciferase and FMN reductase on glass rods.

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