Nitrogen fixation: Cell-free system with extracts of azotobacter

Bulen, William A.; Burns, R. C.; LeComte, Jack R.

doi:10.1016/0006-291x(64)90395-x

Cited by 77 publications

(37 citation statements)

References 9 publications

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“…The nitrogenase was non-particulate, in agreement with reports for nitrogenase of Klebsiella pneumoniae and Clostridium pasteurianum (Ljones, 1974). Particulate nitrogenases have been reported from ' Mycobacterium ' flavum (Biggins & Postgate,I 969) and from Azotobacter vinelandii (Bulen, Burns & LeComte, 1964) although the latter has been questioned by Oppenheim et al (1970) who used osmotic lysis instead of a French pressure cell to disrupt the organism. The soluble nature of Rhizobium nitrogenase is also supported by the rapid release of nitrogenase on incubation of bacteroids with I % Triton X-100; this also released 95 % of the total 3-hydroxybutyrate dehydrogenase activity (de Vries, unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Location of Nitrogenase and Ammonia-assimilatory Enzymes in Bacteroids of Rhizobium leguminosarum and Rhizobium lupini

Planqué¹,

Kennedy²,

Vries³

et al. 1977

Journal of General Microbiology

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95Cytoplasmic and membrane fractions were isolated from bacteroids of Rhizobium leguminosarurn and R . lupini disrupted by lysozyme-induced lysis and sonication. The release of nitrogenase, glutamine synthetase (EC 6 . 3 . I . 2), glutamate synthase (EC 2 . 6 . I .53) and glutamate dehydrogenase (EC I .4. I .4) activities was compared with NADH oxidase and 3-hydroxybutyrate dehydrogenase (EC I . I . I . 30) activities as markers for membrane and cytoplasmic enzyme activities respectively. Nitrogenase was localized in the cytoplasm. Most of the glutamate dehydrogenase activity was released on lysozyme treatment of intact bacteroids although some was released from membrane fractions on sonication, indicating a very superficial localization. Glutamate synthase was present at very low levels in R . Zeguminosarurn bacteroids; the level in R . lupini bacteroids was higher and the enzyme was probably weakly membrane-bound. About 95 % of the glutamine synthetase activity was in the nodule cytosol, and the supernatant of washed bacteroids had a high specific activity; the remaining activity was unevenly distributed between soluble and membrane fractions.

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Section: Discussionmentioning

confidence: 99%

Location of Nitrogenase and Ammonia-assimilatory Enzymes in Bacteroids of Rhizobium leguminosarum and Rhizobium lupini

Planqué¹,

Kennedy²,

Vries³

et al. 1977

Journal of General Microbiology

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“…The latter was reflected by the relative stability to air of the particulate nitrogen-fixing systems extracted from azotobacters (Bulen, Burns & Le Comte, 1964;Kelly, 1966)' compared with the soluble, oxygen-sensitive enzyme fractions obtained on fractionating such particles (Bulen & Le Comte, 1966;Kelly, Klucas & Burris, 1967;Kelly, 1968 b). They suggested that the oxygen-insensitive particle represented a gross manifestation of the ' conformational protection' mechanism against oxygen in Azotobacter.…”

Section: Discussionmentioning

confidence: 99%

“…The particulate character of the system from Mycobacterium JEavum 301 and its failure to utilize pyruvate, recall the system of Azotobacter (Bulen et al 1964;Kelly, I 966) rather than the soluble pyruvate-utilizing system of Clostridium pasteurianum (Carnahan et al 1960).…”

Section: Discussionmentioning

confidence: 99%

Nitrogen Fixation by Cultures and Cell-free Extracts of Mycobacterium flavum 301

Holley

Postgate

1969

Journal of General Microbiology

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S U M M A R YGrowth, nitrogen fixation and acetylene reduction by Mycobacterium Jlavum 301 (NCIB 10,071) were increased with sodium lactate, pyruvate, gluconate or succinate as compared with ethanol, a recommended substrate. Yeast extract could be replaced with (NH4),S04; in continuous culture a source of fixed nitrogen could be omitted altogether. Growth, nitrogen fixation and acetylene reduction all increased at lowered PO, values. Wholly anaerobic conditions did not support growth. Nitrogen fixation was confirmed isotopically.Cell-free extracts performed the following reductions: N, to NH,, Hf to H,, C,H, to C2Hg! KCN to CH,, CH,NC to CH4+C2H,+C2H,. An ATPgenerating system, Mg2-+, Na2S204, and anaerobic conditions during preparation and assay of extracts were required. 3-5 mole ATP were hydrolysed to release I mole H,. Pyruvate, a-ketobutyrate, a-ketoglutarate, succinate, glucose and glucose-6-phosphate did not replace dithionite. ADP, AMP or high coiicentrations of ATP inhibited reduction. Activity was associated with a particle which sedimented at 145,ooog over 3& hr. The nitrogenase system of M . Jlavum thus resembles the particulate system of Azotobacter, rather than the soluble pyruvate-utilizing system of Clostridium pasteurianum.

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“…Nitrogenase acts as a reductant-dependent ATPase [2, 10,11] and one or both of its constituent proteins binds ATP [12,13]. Biggins and Postgate [I41 reported that ATP enhanced the oxygen sensitivity of nitrogenase in crude extracts from the oxygen-sensitive aerobic nitrogen-fixing bacterium Myobacterium flavum 301.…”

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confidence: 99%

The Effect of ATP upon the Oxygen Sensitivity of Nitrogenase from Azotobacter chroococcum

Yates

1972

European Journal of Biochemistry

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1.Crude preparations of nitrogenase (AS1) from Azotobacter chroococcum which were normally oxygen-tolerant, became oxygen-sensitive in the presence of either ATP, GTP, ITP, UTP, CTP or sodium pyrophosphate. MgC1, antagonised this effect of ATY.2. ATP-induced oxygen damage was faster a t 4 "C than a t 30 "C.3. ATP-induced oxygen damage to prepn AS1 affected fraction 2 (Ac2) of the nitrogenase; fraction 1 (Acl) was unaffected. Purified fraction Ac2 became hypersensitive to oxygen in the presence of ATP.4. Pre-treatment of prepn AS1 with "Chelex 100" or EDTA under anaerobic conditions made the nitrogenase oxygen-sensitive. Oxygen-tolerance was partly restored by metal ions. Similar anaerobic pre-treatment of prepn AS1 with ATP followed by removing the ATP by dialysis did not induce oxygen-sensitivity. 5. Pure fraction Ac2 bound significantly more [14C]ATP in the presence of MgCl, than did EDTA-treated fraction Ac2 in the absence of MgC1,.6. Hill plots of the ATP requirement for acetylene reduction, for ATP-induced oxygen damage, for the MgC1, requirement for acetylene reduction and for the MgC1, requirement for protection against oxygen damage to EDTA-treated prepn AS1 all yielded n values between 1 and 2 .7 . These results are discussed in terms of a role for ATP in nitrogen fixation.ATP is an established requirement for nitrogen fixation [l-41 though its role or roles in the reaction have not been determined. It may affect the conformation of the nitrogenase protein [5,6], have a role in electron transfer [7], in substrate binding [8] or in providing a suitable leaving group to form a metal hydride [S].Nitrogenase acts as a reductant-dependent ATPase [2,10,11] and one or both of its constituent proteins binds ATP [12,13]. Biggins and Postgate [I41 reported that ATP enhanced the oxygen sensitivity of nitrogenase in crude extracts from the oxygen-sensitive aerobic nitrogen-fixing bacterium Myobacterium flavum 301. The present paper describe a study of a similar effect of ATP upon the nitrogenase from Azotobacter chroococcum, suggests how it is caused and a possible role for ATP in nitrogen fixation. The growth, harvesting and partial purification of nitrogenase from A . chroococcum was described previously [15,16]. The further purification of the nitrogenase into its component fractions (designated Acl and Ac2 for the Mo-Fe protein and the Fe protein respectively) will be described elsewhere. MATERIALS AND METHODS BiochemicalsCrude supernatants (prepn AS1) were obtained by disrupting A. chroococcum in a French pressure cell from 3000 lb/in2 and centrifuging the suspension a t 400OOxg for 25 min to remove unbroken cells and cell debris. This supernatant is comparable to the previously reported particulate preparations from Azotobacter [3,18], and contained magnesium, calcium, iron, molybdate and zinc ions a t 200, 12, 23, 0.4 and 0.5 ng atoms/mg protein, respectively; mangenese, copper and cobalt were not detected. Further purification rendered the nitrogenase oxygen-sensitive and therefore unsuitable for the experi...

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Nitrogen fixation: Cell-free system with extracts of azotobacter

Cited by 77 publications

References 9 publications

Location of Nitrogenase and Ammonia-assimilatory Enzymes in Bacteroids of Rhizobium leguminosarum and Rhizobium lupini

Location of Nitrogenase and Ammonia-assimilatory Enzymes in Bacteroids of Rhizobium leguminosarum and Rhizobium lupini

Nitrogen Fixation by Cultures and Cell-free Extracts of Mycobacterium flavum 301

The Effect of ATP upon the Oxygen Sensitivity of Nitrogenase from Azotobacter chroococcum

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