Detection of nitrogenase components and other nonheme iron proteins in polyacrylamide gels

Brill, Winston J.; Westphal, Josh; Stieghorst, Michael F.; Davis, Lawrence C.; Shah, Vinod K.

doi:10.1016/0003-2697(74)90149-3

Cited by 35 publications

(7 citation statements)

References 5 publications

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“…Suzuki et al (16) reported that a Figure 1. The gel was stained with Coomassie brilliant blue (A) or with a,a-dipyridyl (B) as described in (2), or analyzed by immunoblotting with an antiserum against Fd I (C). Lane We have studied changes in the relative abundance of Fd isoproteins during growth of seedlings and the effect of illumination on the level of each isoprotein.…”

Section: Resultsmentioning

confidence: 99%

“…A slab gel (13.5 x 15 x 0.1 cm) was prepared using linear gradient of acrylamide from 15 to 25%. All electrtophoretic separations were performed in a cold chamber, at 4°C with prechilled buffer and gel at a constant current of 20 mamp for 2.5 h. Gels were stained with Coomassie brilliant blue R-250 and if necessary, proteins containing non-heme iron were visualized with a,a-dipyridyl and thioglycolate as described by Brill et al (2). For Western blot analysis of the electrophoresis gel using a rabbit antibody raised against the purified Fd I, proteins were electrophoretically transferred to Immobilon (Millipore) after the gel is boiled for 10 min to denature Fd, and the polypeptides bound by the antibody are visualized with '25I-protein A.…”

Section: Electrophoresis and Western Blotmentioning

confidence: 99%

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Localization of Ferredoxin Isoproteins in Mesophyll and Bundle Sheath Cells in Maize Leaf

Kimata

Hase²

1989

Plant Physiol.

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MATERIALS AND METHODS Four ferredoxin isoproteins were identifed in the C4 plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etfolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of effolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C4 plant.The presence of different molecular species of plant-type Fd in one organism has been reported in a variety of higher plants and algae (4,7,11,15,17), and variations in the Fd isoproteins with stages and conditions of growth have been observed (4,17,20). Although several attempts have been made to examine whether the Fd isoproteins have different functions in different aspects of Fd-linked metabolism, no conclusive evidence has been obtained for the biological significance ofthe isoproteins (4,7,19). Recently, the heterocyst of a blue-green alga, Anabaena variabilis, was shown to contain a specific Fd which is distinct from the Fd found in vegetative cells. The heterocyst Fd seemed to be involved in a specific interaction with nitrogenase (15).In the leaves of C4 plants, the metabolic process of photosynthesis is partitioned between two types of cell, BSC' and MC, and the intercellular compartmentalization ofthe assimilation of carbon and nitrogen is well documented (5). Furthermore, in the leaves of NADP-malic enzyme types of species, such as maize, almost all of capacity for non-cyclic electron flow is localized in the MC, and the PSII activity of the chloroplasts in the BSC is weak (5). In the course of a survey of Fd isoproteins in C4 plants, we Extraction and Partial Purification of FdDark-grown seedlings were divided into three parts, leaves including coleoptiles, mesocotyls and primary roots. Mesocotyl is the first internodal part formed above the seed storage tissues, which elongates considerably during growth in darkness. Ten g each of these tissues were ground with a mortar and pestle with 1 g of polyclar AT and a small amount of quartz sand in 40 ml of ice-cold extraction buffer (20 mM Tris-HCl (pH 7.5), 100 mm NaCl, 1 mM MgCl2, 1 mM EDTA, 1 mM PMSF, and 1%[v/v] 2-mercaptoethanol). The homogenate was filtered through two layers of cheese cloth and centrifuged at 12,000g for 10 min. The resulting supernatant was mixed with an excess amount of DEAE-cellulose (about 20 g wet weight) and the suspension of resin was packed in a column. After the column wa...

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Section: Resultsmentioning

confidence: 99%

Section: Electrophoresis and Western Blotmentioning

confidence: 99%

Localization of Ferredoxin Isoproteins in Mesophyll and Bundle Sheath Cells in Maize Leaf

Kimata

Hase²

1989

Plant Physiol.

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“…This protein appeared to be synthesized in low amounts and could not be detected by its colour until the penultimate step of purification, an anion-exchange chromatography, which separated it from the abundant dark-brown FdI. Analysis of the pink fraction by electrophoresis on a non-denaturing polyacrylamide gel showed a coloured band which reacted positively when the gel was revealed with a stain specific for the detection of non-heme iron in proteins (Brill et al, 1974). This suggested that the fraction contained an ironsulfur protein.…”

Section: Identification and Purification Of A New Ferredoxin From R mentioning

confidence: 98%

Purification of a sixth ferredoxin from Rhodobacter capsulatus

Naud¹,

Vincon

Garin

et al. 1994

European Journal of Biochemistry

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A new ferredoxin has been purified from the photosynthetic bacterium Rhodobacter capsulatus. It is the sixth ferredoxin to be isolated from this bacterium and it was called FdVI.Its primary structure was established based on amino acid sequence analysis of the protein and of peptides derived from it. It is composed of 106 residues including five cysteines. The calculated mass of the polypeptide is 11402.6 Da which matches the experimental value determined by electrospray mass spectrometry. Amino acid sequence comparison revealed that ferredoxin VI (FdVI) is strikingly similar to a ferredoxin from Caulobacter crescentus and to the putidaredoxin from Pseudomonas putida.FdVI exhibited an ultraviolet-visible absorption spectrum typical for a [2Fe-2S] ferredoxin. EPR spectroscopy of the reduced protein showed a nearly axial signal similar to that of mitochondria1 and €? putida ferredoxins.FdVI is biosynthesized in cells growing anaerobically under either nitrogen-sufficient or nitrogen-deficient conditions. Although the function of FdVI is unknown, its structural resemblance to [2Fe-2S] ferredoxins known to transfer electrons to oxygenases such as P-450 cytochromes, suggests that FdVI may have a similar role in R. capsulatus.The photosynthetic bacterium Rhodobacter capsulatus is a facultative phototroph endowed with remarkable abilities of metabolic adaptation. It can grow autotrophically or heterotrophically either in the light or in darkness and can choose between five different growth modes (Madigan and Gest, 1979). It is also capable of fixing molecular nitrogen through a metabolic process which has been extensively studied by biochemical (Hallenbeck et al., 1982a; and genetic approaches (Willison et al., 1985;Klipp et al., 1988). Nitrogen fixation is catalyzed by nitrogenase and requires ATP and a low-potential reductant. Two types of electron-carrier proteins, ferredoxins (Fd) and flavodoxins, are known to serve as electron donors to nitrogenase. In R. capsulatus, the identification of the actual physiological reductant of nitrogenase is complicated by the occurence in this bacterium of an exceptional diversity of electron carriers. Five distinct ferredoxins have been isolated and biochemically characterized; three of them, FdI, FdII and FdIII, appear as representative molecular forms of the dicluster ferredoxins found in a wide range of bacteria (Bruschi and Guerlesquin, 1988). FdI and the homodimeric FdIII, both contain two [4Fe-4S] clusters/monomer (Hallenbeck et al.,

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“…The FeMoCo, which is visible as a dark brown band, was eluted in 1.5 ml of 65 mM Tris-HCl buffer, pH 7.4, in about 2.5 hr from the time it was applied to the gel. Analytical acrylamide gel electrophoresis was carried out anaerobically (22) and stained for protein or Fe as described (27).…”

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confidence: 99%