Activation of L‐ornithine by cell‐free extracts of <i>Bacillus brevis</i> catalyzing the synthesis of gramicidin S

Sand, Tor‐Erik; Siebke, J. C.; Laland, S.

doi:10.1016/0014-5793(68)80019-5

Cited by 8 publications

(3 citation statements)

References 15 publications

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“…The next stage, naturally, is the isolation of the individual enzymes and the elucidation of the mechanism of synthesis. Recently, fractionation of the gramicidin S synthesizing activity from an 11,000~g supernatant of B. brevis was briefly described [4]. The present report is a more detailed account of and also an extension of these experiments.…”

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confidence: 77%

Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts

Bredesen¹,

Berg²,

Figenschou³

et al. 1968

European Journal of Biochemistry

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A 20 fold purification of the gramicidin S synthesizing activity in a 11,OOOxg supernatant from Bacillus brevis has been achieved. The purification procedure involves precipitation of undesired material with streptomycin sulphate, followed by precipitation of gramicidin S synthesizing activity a t 40°/, (NH,),SO, saturation and fractionation on DEAE-Sephadex. All gramicidin S synthesizing activity appeared in one peak. It incorporated labelled phenylalanine, proline, valine, ornithine and leucine to the same extent into gramicidin S. The gramicidin S synthesizing activity was found to be stable in a 0.05 M potassium phosphate buffer (pH 7.1) containing glutathione (0.01 M) and glycerol (20°/,,, v/v) for a t least 3 days a t + 4" and four weeks at -20".Experiments with cell-free extracts from Bacillus brevis has established that gramicidin S is synthesized by a mechanism which is different from that of proteins [l-31. The next stage, naturally, is the isolation of the individual enzymes and the elucidation of the mechanism of synthesis. Recently, fractionation of the gramicidin S synthesizing activity from an 11,000~g supernatant of B. brevis was briefly described [4]. The present report is a more detailed account of and also an extension of these experiments. Since the gramicidin S synthesizing activity was found to be unstable, attempts were made to stabilize it. It was found that the gramicidin S synthesizing activity was stable a t $-4" in the presence of glycerol, glutathione (GSH) and phosphate buffer. Purification of the gramicidin S synthesizing activity from an 11,000 x g supernatant using streptomycin sulphate, (NH,),SO, and fractionation on a DEAE-Sephadex column resulted in a 20 fold purification with the activity in one peak. MATERIALS AND METHODS Growth of the OrganismThe Bacillus brevis strain ATCC 9999 was grown under aeration (5 1 airlmin) in 10 1 of the synthetic medium described by Eikhom, Jonsen, Laland

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mentioning

confidence: 77%

Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts

Bredesen¹,

Berg²,

Figenschou³

et al. 1968

European Journal of Biochemistry

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“…A number of procedures have been described for the measurement of this activity [1][2][3][4]. They differ slightly in their experimental conditions, but all require the adsorption of the labelled ATP on activated charcoal after incubation in the presence of labelled PPi-The labelled ATP was then measured directly [1,2] on the charcoal or after elution with 1 N HCL [3] or by NH4OH [4] and measuring an aliquot of the solution in a liquid scintillation counter.…”

Section: E-amp + Aa ~-E-aa ÷ Amp (2)mentioning

confidence: 99%

Amino acid dependent ATP—³²PP_i exchange measurement a filter paper disk method

Simlot¹,

Pfaender²

1973

FEBS Letters

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“…Gramicidin S (GS) synthetase is an enzyme complex responsible for the nonribosomal biosynthesis of GS in Bacillus brevis. The complex includes both a heavy (280,000 daltons) and a light (100,000 daltons) enzyme and catalyzes the formation of the cyclic decapeptide GS: 2 In the first step of GS biosynthesis, the individual amino acids are activated by ATP, which results in the formation of aminoacyl adenylates (7,12,21). The light enzyme catalyzes activation and racemization of L-to Dphenylalanine, and the heavy enzyme activates I Presnt address: George Washington University Law School, Washington, DC 20003. the other constituent amino acids (10).…”

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confidence: 99%

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis

Friebel¹,

Demain²

1977

J Bacteriol

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Incorporation of L_[14C]ornithine into gramicidin S by crude, unfractionated lysozyme extracts of Bacillus brevis ATCC 9999 was shown to represent the activity of the gramicidin synthetase complex. Frozen-thawed cells were the source of active extracts, but when cells were shaken in air at 370C, they rapidly lost activity in a first-order reaction with a half-life of 13 min. Protease inhibitors and inhibitors of energy metabolism had no effect on the inactivation process in frozen-thawed cells. Stabilization was achieved when the cells were shaken in nitrogen or helium instead of air. The addition of dithiothreitol produced a moderate degree of stabilization. The i-ornithineand i-phenylalanine-activating activities of the gramicidin S synthetase complex were also lost during aeration of the cells. Crude cell-free extracts also lost activity when they were shaken in oxygen, but, in this case, inactivation was slower (half-life of 80 min). Nitrogen also stabilized these cell-free extracts. 1010 on August 1, 2020 by guest

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Activation of L‐ornithine by cell‐free extracts of Bacillus brevis catalyzing the synthesis of gramicidin S

Cited by 8 publications

References 15 publications

Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts

Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts

Amino acid dependent ATP—³²PP_i exchange measurement a filter paper disk method

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis

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Activation of L‐ornithine by cell‐free extracts of Bacillus brevis catalyzing the synthesis of gramicidin S

Cited by 8 publications

References 15 publications

Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts

Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts

Amino acid dependent ATP—32PPi exchange measurement a filter paper disk method

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis

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Amino acid dependent ATP—³²PP_i exchange measurement a filter paper disk method