On the Role of Menaquinone in the Reduced Nicotinamide Adenine Dinucleotide Oxidative Pathway of Bacillus brevis

Fynn, G. H.; Thomas, Dayon; Seddon, B.

doi:10.1099/00221287-70-2-271

Cited by 7 publications

(5 citation statements)

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“…We have recently characterized the electron-transport system of this strain and have identified a menaquinone (MK-7) by thin-layer chromatography and by U.V. and mass spectroscopy to be the sole quinone component (Fynn, Thomas & Seddon, 1972). Pentane extraction (Szarkowska, I 966) and acetone extraction (Fynn & Redfearn, 1964) were used to remove the endogenous quinone in an attempt to confirm a functional role for MK-7 in the electron transport system.…”

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Menaquinone Function in the Electron Transport System of an Antibiotic-producing Strain of Bacillus brevis

Fynn

1973

Journal of General Microbiology

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Bacillus brevis ATCC I 0068 grown under appropriate growth conditions produces a mixture of tyrocidine and gramicidin, which are potent inhibitors of electron transport and oxidative phosphorylation processes. We have recently characterized the electron-transport system of this strain and have identified a menaquinone (MK-7) by thin-layer chromatography and by U.V. and mass spectroscopy to be the sole quinone component (Fynn, Thomas & Seddon, 1972). Pentane extraction (Szarkowska, I 966) and acetone extraction (Fynn & Redfearn, 1964) were used to remove the endogenous quinone in an attempt to confirm a functional role for MK-7 in the electron transport system. Although these methods were successful in removing 90 to 95 % of the quinone and largely abolishing the NADH oxidase activity of the preparation, the addition of MK-7 in various carrier systems failed to restore the latter activity. However, from the literature it is clear that while restoration of succinoxidase activity of acetone-extracted preparations has been successfully demonstrated, the reactivation of the NADH oxidase pathway has been more difficult to achieve. Also the pentane extraction technique suffers from the disadvantage that it can only be successfully employed to extract lyophilized material. The lyophilization process itself involves some structural damage to the electron transport particle since the NADH oxidase activity falls to 40 to 60 % of its original value. We now report on the U.V. irradiation of fresh material as a means of inactivating the endogenous menaquinone and providing the basis for a further investigation of quinone function in the organism. METHODS U.V. irradiation. Irradiation with 364 nm light was most effective in abolishing the NADH oxidase activity of the freshly prepared 105 P material : shorter wavelength (254 nm) was less effective and produced a general inactivation of the enzyme activities of the preparation. Irradiation was carried out with a 125 W Phillips 57236 F/70 mercury arc source placed at a distance of 10 cm above a Petri dish containing a 3 m~ depth of 105 P preparation in tris-HC1 buffer (0.1 M, p H 7.4). The preparation was stirred on ice by means of a magnetic stirrer and kept covered with a borosilicate glass plate to avoid evaporation losses.Quinone/phospholipid rnicelles. The appropriate phospholipid (see Results) to give a final concentration of 15 mg/ml was dissolved in a minimum volume of chloroform and the calculated volume of a I O -~ M-ethanolic solution of quinone added. The mixture was evaporated to dryness on a rotary evaporator at 20 "C and resuspended in 0.01 M, pH 7.8, tris-EDTA buffer. The suspension was sonicated for 15 min at o "C using a MSE-Mullard type 4200 60 W ultrasonic disintegrator fitted with a stainless-steel vibrator probe of 6 mm end diameter and 9: I end ratio. The sonicated material was centrifuged at Iooooog for 45 min and any insoluble residue discarded.

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Menaquinone Function in the Electron Transport System of an Antibiotic-producing Strain of Bacillus brevis

Fynn

1973

Journal of General Microbiology

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“…However, in the case of NADH oxidase activity of Bacillus brevis, no restoration was observed upon addition of the natural quinone (Fynn, Thomas & Seddon, 1972). The low level of restoration of H,:fumarate reductase activity of u.v.-irradiated D. gigas particles may be due to failure of the techniques used to place MK-6 back to its original site in the particle (Fynn et al 1972). In particular, the presence of altered endogenous menaquinone in irradiated D. gigas-particles could prevent reincorporation of exogenously added quinone (Knook & Planta, 1971).…”

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confidence: 93%

“…Similar results, corresponding to a partial restoration of enzymic activities by quinone reincorporation into extracts depleted of these carriers, have already been reported (Knook & Planta, 1971 ;Kroger & Dadak, 1969). However, in the case of NADH oxidase activity of Bacillus brevis, no restoration was observed upon addition of the natural quinone (Fynn, Thomas & Seddon, 1972). The low level of restoration of H,:fumarate reductase activity of u.v.-irradiated D. gigas particles may be due to failure of the techniques used to place MK-6 back to its original site in the particle (Fynn et al 1972).…”

Section: Short Cornmuti Ica T Ionmentioning

confidence: 99%

On the Role of Menaquinone-6 in the Electron Transport of Hydrogen: Fumarate Reductase System in the Strict Anaerobe Desulfovibrio gigas

Hatchikian

2000

Microbiology

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“…Bacillus brevis ATCC I 0068 was maintained and grown as described previously . The glycerol-asparagine (GA) medium was replaced by a peptone-yeast extract (PYE) medium (Fynn, Thomas & Seddon, 1972) for some studies. The dissolved 0, concentration of the bacterial culture was measured by means of a submerged Clark-type 0, electrode fitted into the incubation flask under aseptic conditions (oxygen content is quoted as percentage air-saturated media at 37 "C).…”

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Energetics of Growth in a Tyrothricin-producing Strain of Bacillus brevis

Seddon

Fynn

1973

Journal of General Microbiology

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S U M M A R YThe particulate NADH oxidase activity of Bacillus brevis is inhibited by tyrocidine and the kinetics of inhibition indicate that the NADH dehydrogenase region is the primary site of action. A decrease in NADH oxidase activity of electron transport particles and of NADH oxidase activity of whole organisms occurs in batch culture in the later stages of growth when production of tyrothricin increases. That respiration exhibited by growing cells takes place via the respiratory electrontransport system is indicated by the similarities in levels of activity and cyanide sensitivity of endogenous and NADH oxidation of growing cells. Exponential growth on glycerol asparagine media is diphasic, a slower growth rate commencing as the 0, tension of the culture approaches zero and tyrothricin production increases. Molar growth yield data based on overall measurements of growth related to 0, uptake in batch culture lead to low ATP/O ratios. In an attempt to determine Yo and ATP/O ratios under optimum conditions of growth a method is described which was based entirely on the growth and respiratory activity of exponentially growing cells. Such measurements led to higher values for Yo and to ATP/O ratios approaching 3, indicating the participation of three sites of phosphorylation during exponential growth. Tyrocidine and DNP inhibited growth, the tyrocidine at concentrations similar to those produced at the end of the exponential growth phase. Yo/Yl,, ratios (ATP/O ratios) on glycerol asparagine media were lower than on peptone yeast extract medium which yielded no tyrothricin, a possible indication that some form of uncoupling occurs in organisms grown on glycerol asparagine medium which does not yield tyrothricin.

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On the Role of Menaquinone in the Reduced Nicotinamide Adenine Dinucleotide Oxidative Pathway of Bacillus brevis

Cited by 7 publications

References 15 publications

Menaquinone Function in the Electron Transport System of an Antibiotic-producing Strain of Bacillus brevis

Menaquinone Function in the Electron Transport System of an Antibiotic-producing Strain of Bacillus brevis

On the Role of Menaquinone-6 in the Electron Transport of Hydrogen: Fumarate Reductase System in the Strict Anaerobe Desulfovibrio gigas

Energetics of Growth in a Tyrothricin-producing Strain of Bacillus brevis

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