Characterization of two Cu-containing protein fragments obtained by limited proteolysis of Hyphomicrobiumdenitrificans A3151 nitrite reductase

Abstract In a previous report (Blood 60:265, 1982), we described a family with an abnormal RBC membrane protein doublet, which we considered a shortened protein 4.1 on the basis of biochemical and genetic data. Using an anti-4.1 monoclonal antibody, we confirm here that the shortened protein derives from protein 4.1. One of the members of the family contemporaneously displayed the 4.1 (-) trait, eg, the heterozygous state of this variety of hereditary elliptocytosis that lacks protein 4.1. The 4.1a/4.1b ratio was low whenever the 4.1 trait was present, regardless of the type of protein 4.1 involved. The RBCs of the compound heterozygote, containing only the shortened species of protein 4.1, made it possible to analyze without interference the contact between shortened protein 4.1 and sialoglycoprotein beta, or glycoconnectin. Shortened protein 4.1 did not alter the amount of glycoconnectin in the ghosts nor did it change its extractability into the Triton shells. Limited proteolysis of shortened polypeptides 4.1a and 4.1b showed that they are sequence related. It is conflicting that the persons carrying the shortened protein 4.1 are devoid of specific clinical and morphological abnormalities, apart from those pertaining to the 4.1- trait, when the latter is present.

Download Full-text

The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of the nirAGene and Characterization of the Recombinant Enzyme

Journal of Bacteriology ◽

10.1128/jb.181.8.2323-2329.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2323-2329 ◽

Cited By ~ 25

Author(s):

Miguel Prudêncio ◽

Robert R. Eady ◽

Gary Sawers

Keyword(s):

Amino Acid ◽

Nitrite Reductase ◽

Recombinant Enzyme ◽

Leader Peptide ◽

Resonance Spectroscopy ◽

Gene Encoding ◽

Blue Copper

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Download Full-text

Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k)

Planta ◽

10.1007/s004250000327 ◽

2000 ◽

Vol 211 (3) ◽

pp. 440-445 ◽

Cited By ~ 13

Author(s):

Nicole Burhenne ◽

Rudolf Tischner

Keyword(s):

Nitrite Reductase ◽

Chlorella Sorokiniana ◽

Isolation And Characterization

Download Full-text

Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by X-ray diffraction and electron microscopy

Journal of Molecular Biology ◽

10.1016/0022-2836(80)90272-7 ◽

1980 ◽

Vol 140 (4) ◽

pp. 581

Keyword(s):

Electron Microscopy ◽

Pseudomonas Aeruginosa ◽

Cytochrome Oxidase ◽

Nitrite Reductase ◽

X Ray Diffraction ◽

X Ray

Download Full-text

Purification and Characterization of a Dissimilatory Nitrite Reductase from the Phototrophic Bacterium Rhodopseudomonas palustris

Zeitschrift für Naturforschung C ◽

10.1515/znc-1983-11-1210 ◽

1983 ◽

Vol 38 (11-12) ◽

pp. 933-938 ◽

Cited By ~ 10

Author(s):

Michaela Preuß ◽

Jobst-Heinrich Klemme

Keyword(s):

Cytochrome C ◽

Nitrite Reductase ◽

Rhodopseudomonas Palustris ◽

Gel Filtration ◽

Enzyme Synthesis ◽

Nitrite Reduction ◽

Phototrophic Bacterium ◽

Dissimilatory Nitrite Reductase ◽

Basic Level

A dissimilatory nitrite reductase from the facultatively phototrophic bacterium , Rhodopseudomonas palustris strain 1a1 was studied. A basic level of the enzyme (10 -50 mU/mg protein) was measured in dark, aerated and anaerobic, photosynthetic cultures. A marked derepression of enzyme synthesis occurred under conditions of oxygen limitation (200-300 mU/mg protein). The addition of nitrite (or nitrate) to the culture medium had only a slight effect on the maximal nitrite reductase titer of cells. The enzyme was purified from photosynthetically grown cells by precipitation with ammonium sulfate, gel filtration through Sepharose 6B and repeated chromatography on DE 52-cellulose. As estimated by gel filtration, the nitrite reductase had a molecular weight of about 120 000 ± 12 000 and yielded only one band (mol. wt. of about 68 000 ± 7000) in SDS-gel electrophoresis. The isoelectric point of the enzyme was at pH 5.1. Nitric oxide (NO) was identified as the reaction product of nitrite reduction. The enzyme also exhibited cytochrome c-oxidase activity and was active with chemically reduced viologen dyes, FMN and cytochrome c as electron donors. Highly purified nitrite reductase preparations contained 10 mol% of a c-type cytochrome. Trace metal analyses indicated the presence of Cu in the enzyme. Consistent with the detection of Cu was the finding that the Cu-chelator, diethyldithiocarbamate, strongly inhibited the nitrite reductase

Download Full-text