Quaternary structure of assimilatory NADH:nitrate reductase from Chlorella.

Howard, William D.; Solomonson, L P

doi:10.1016/s0021-9258(18)34011-0

Cited by 52 publications

(9 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Succinate dehydrogenase was purified from bovine heart mitochondria as described by Baginsky & Hatefi (1969). NADH-linked nitrate reductase was purified to electrophoretic homogeneity from Chlorella vulgaris as previously described (Howard & Solomonson, 1982). Xanthine oxidase was isolated from bovine milk as described by Barber & Siegel (1982).…”

Section: Methodsmentioning

confidence: 99%

Anti-flavin antibodies

Barber¹,

Eichler²,

Solomonson³

et al. 1987

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.

show abstract

Section: Methodsmentioning

confidence: 99%

Anti-flavin antibodies

Barber¹,

Eichler²,

Solomonson³

et al. 1987

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…Although in these different organisms it catalyzes a similar reduction of nitrate involving NADH or NADPH as electron donor, the enzyme displays a variety of structures and dissimilarities. In Escherichia coli the enzyme is an heterotrimer bound to the bacterial membrane (Rasul Chaudhry and MacGregor 1953) and in ChloreIla NR is an homotetramer (Howard and Solomonson 1982) found in the pyrenoid. In plants the enzyme is an homodimer possibly interacting with the chloroplast outer membrane.…”

Section: Introductionmentioning

confidence: 99%

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants

et al. 1987

View full text Add to dashboard Cite

Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the -galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

show abstract

“…

b-type cytochrome, and Mo-pterin prosthetic groups in a ratio of 1:1:1 (Howard & Solomonson, 1982). The strong visible absorbance of the b-type cytochrome of NR has enabled the redox properties of this center to be studied in detail (Kay 1 Abbreviations: NR, nitrate reductase; EPR, electron paramagnetic resonance; EDTA, ethylenediaminetetraacetic acid; CD, circular dichroism; CIP, corn inactivating protease; MV,+, methylviologen radical; FAD-", flavin adenine dinucleotide semiquinone anion; FADH™, flavin adenine dinucleotide hydroquinone.

…”

mentioning

confidence: 99%

CD and potentiometry of FAD, heme and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase

Kay

Barber²,

Solomonson³

1988

Biochemistry

View full text Add to dashboard Cite

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Quaternary structure of assimilatory NADH:nitrate reductase from Chlorella.

Cited by 52 publications

References 47 publications

Anti-flavin antibodies

Anti-flavin antibodies

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants

CD and potentiometry of FAD, heme and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase

Contact Info

Product

Resources

About