scholarly journals Purification and characterization of assimilatory nitrite reductase from Candida utilis

1996 ◽  
Vol 317 (1) ◽  
pp. 147-155 ◽  
Author(s):  
Sagar SENGUPTA ◽  
Melkote S. SHAILA ◽  
Gannamani R. RAO
Keyword(s):  
Nitrite Reductase ◽  
Candida Utilis ◽  
Gel Filtration ◽  
Nitrate Assimilation ◽  
Nitrite Reduction ◽  
In Vitro Translation ◽  
Nitrite Reductases ◽  
Stepwise Reduction ◽  
Molecular Masses

Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identity of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

New insights into the activity of Pseudomonas aeruginosa cd1 nitrite reductase

2008 ◽  
Vol 36 (6) ◽  
pp. 1155-1159 ◽  
Author(s):  
Serena Rinaldo ◽  
Alessandro Arcovito ◽  
Giorgio Giardina ◽  
Nicoletta Castiglione ◽  
Maurizio Brunori ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Nitrite Reduction ◽  
Nitrite Reductases ◽  
Cytochrome Cd1 ◽  
Haem Iron ◽  
Shed Light ◽  
Denitrification Process

The cytochrome cd1 nitrite reductases are enzymes that catalyse the reduction of nitrite to nitric oxide (NO) in the bacterial energy conversion denitrification process. These enzymes contain two different redox centres: one covalently bound c-haem, which is reduced by external donors, and one peculiar d1-haem, where catalysis occurs. In the present paper, we summarize the current understanding of the reaction of nitrite reduction in the light of the most recent results on the enzyme from Pseudomonas aeruginosa and discuss the differences between enzymes from different organisms. We have evidence that release of NO from the ferrous d1-haem occurs rapidly enough to be fully compatible with the turnover, in contrast with previous hypotheses, and that the substrate nitrite is able to displace NO from the d1-haem iron. These results shed light on the mechanistic details of the activity of cd1 nitrite reductases and on the biological role of the d1-haem, whose presence in this class of enzymes has to date been unexplained.

The human L-myc gene encodes multiple nuclear phosphoproteins from alternatively processed mRNAs

1988 ◽  
Vol 8 (10) ◽  
pp. 4381-4388
Author(s):  
J De Greve ◽  
J Battey ◽  
J Fedorko ◽  
M Birrer ◽  
G Evan ◽  
...  
Keyword(s):  
Rna Processing ◽  
In Vitro Translation ◽  
Intron 1 ◽  
Myc Gene ◽  
Molecular Masses ◽  
Steady State Levels ◽  
Matrix Fraction ◽  
Nuclear Phosphoproteins

The human proto-oncogene L-myc generates at least four different mRNAs by alternative RNA processing. We have identified two phosphorylated L-myc proteins with molecular masses of 60,000 and 66,000 daltons [p60L-myc(human) and p66L-myc(human)] in a small-cell carcinoma line expressing high levels of L-myc mRNA. These proteins have a short half-life and are localized to the nuclear matrix fraction, as previously reported for the c-myc and N-myc proteins. In vitro translation experiments demonstrated that both the p60 and p66 species are encoded by a 3.9-kilobase (kb) mRNA which retains intron 1, while only the p60 protein is translated from a 3.6-kb L-myc mRNA which has had intron 1 removed. While L-myc proteins [p32L-myc(human) and p37L-myc(human)] could be synthesized in vitro from 2.2-kb mRNA templates, no such proteins were detected by immunoprecipitation in vivo. These observations suggest that alternative RNA processing of the L-myc transcript could play a role in determining the steady-state levels of the p60L-myc and p66L-myc proteins.

scholarly journals Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics ofIgnicoccusSpecies

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Lydia J. Kreuter ◽  
Andrea Weinfurtner ◽  
Alexander Ziegler ◽  
Julia Weigl ◽  
Jan Hoffmann ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Cellular Membrane ◽  
Content Type ◽  
The Pacific ◽  
Native Electrophoresis ◽  
Molecular Masses ◽  
The Pacific Ocean

ABSTRACTIn this study, the ATP synthase ofIgnicoccus hospitaliswas purified, characterized, and structurally compared to the respective enzymes of the otherIgnicoccusspecies, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genusIgnicoccuscomprises three described species, i.e.,I. hospitalisandIgnicoccus islandicusfrom hot marine sediments near Iceland andIgnicoccus pacificusfrom a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC).I. hospitalisis the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeonNanoarchaeum equitans.I. hospitalisgrows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome ofI. hospitalisencodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximalin vitroactivity of theI. hospitalisenzyme was measured around pH 6, the optimal stability of the A1AOcomplex seemed to be at pH 9. Interestingly, the soluble A1subcomplexes of the differentIgnicoccusspecies exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCETheCrenarchaeotarepresent one of the major phyla within theArchaeadomain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of theCrenarchaeotauntil now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subjectI. hospitalishas a particular importance among crenarchaeotes, since it is the only known host ofN. equitans. The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

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

1983 ◽  
Vol 38 (11-12) ◽  
pp. 933-938 ◽  
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

scholarly journals Post-translational processing of the phosphatidylserine decarboxylase gene product in Chinese hamster ovary cells

1996 ◽  
Vol 319 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Osamu KUGE ◽  
Kyoko SAITO ◽  
Michiyuki KOJIMA ◽  
Yuzuru AKAMATSU ◽  
Masahiro NISHIJIMA
Keyword(s):  
Gene Product ◽  
Cdna Clone ◽  
Chinese Hamster Ovary ◽  
Cdna Sequence ◽  
Chinese Hamster ◽  
In Vitro Translation ◽  
Decarboxylase Activity ◽  
Short Period ◽  
Molecular Masses

We have isolated a full-length cDNA clone of the Chinese hamster ovary (CHO) pssC gene, which encodes mitochondrial phosphatidylserine decarboxylase. The cDNA clone is capable of increasing phosphatidylserine decarboxylase activity to 11-fold in CHO-K1 cells. The pssC gene product predicted from the cDNA sequence is composed of 409 amino acid residues. In an in vitro translation system coupled with in vitro transcription, the cDNA clone directs the formation of a protein with an apparent molecular mass of 46 kDa. In CHO-K1 cells, the cDNA clone leads to the production of two major peptides with apparent molecular masses of 38 and 34 kDa, as determined by Western blotting with an antibody raised against a recombinant pssC protein. When CHO-K1 cells transfected with the cDNA clone are labelled with [35S]methionine for a short period, proteins immunoprecipitated with the antibody lack radioactive 38 and 34 kDa peptides, but contain two radioactive peptides with apparent molecular masses of 46 and 42 kDa instead. The pssC gene product predicted from the cDNA sequence has, near its C-terminus, a unique Leu-Gly-Ser-Thr sequence which is known as a processing site for Escherichia coli phosphatidylserine decarboxylase. A mutant pssC cDNA clone, in which Ser378 in the conserved sequence is replaced by Ala, leads to overproduction of 46, 42 and 38 kDa peptides, but not a 34 kDa peptide. This mutant clone is incapable of increasing phosphatidylserine decarboxylase activity, in contrast to the wild-type clone. These results indicate that the processing at the Leu-Gly-Ser-Thr sequence is essential for formation of the active enzyme. Thus, the pssC gene product is converted into mature phosphatidylserine decarboxylase through multiple steps of post-translational processing.

scholarly journals The human L-myc gene encodes multiple nuclear phosphoproteins from alternatively processed mRNAs.

1988 ◽  
Vol 8 (10) ◽  
pp. 4381-4388 ◽  
Author(s):  
J De Greve ◽  
J Battey ◽  
J Fedorko ◽  
M Birrer ◽  
G Evan ◽  
...  
Keyword(s):  
Rna Processing ◽  
In Vitro Translation ◽  
Intron 1 ◽  
Myc Gene ◽  
Molecular Masses ◽  
Steady State Levels ◽  
Matrix Fraction ◽  
Nuclear Phosphoproteins

The human proto-oncogene L-myc generates at least four different mRNAs by alternative RNA processing. We have identified two phosphorylated L-myc proteins with molecular masses of 60,000 and 66,000 daltons [p60L-myc(human) and p66L-myc(human)] in a small-cell carcinoma line expressing high levels of L-myc mRNA. These proteins have a short half-life and are localized to the nuclear matrix fraction, as previously reported for the c-myc and N-myc proteins. In vitro translation experiments demonstrated that both the p60 and p66 species are encoded by a 3.9-kilobase (kb) mRNA which retains intron 1, while only the p60 protein is translated from a 3.6-kb L-myc mRNA which has had intron 1 removed. While L-myc proteins [p32L-myc(human) and p37L-myc(human)] could be synthesized in vitro from 2.2-kb mRNA templates, no such proteins were detected by immunoprecipitation in vivo. These observations suggest that alternative RNA processing of the L-myc transcript could play a role in determining the steady-state levels of the p60L-myc and p66L-myc proteins.

scholarly journals Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules.

1990 ◽  
Vol 111 (4) ◽  
pp. 1363-1371 ◽  
Author(s):  
M Zanetti ◽  
L Litteri ◽  
R Gennaro ◽  
H Horstmann ◽  
D Romeo
Keyword(s):  
Bone Marrow Cells ◽  
In Vitro Translation ◽  
Bovine Bone ◽  
The Matrix ◽  
Molecular Masses ◽  
Triton X ◽  
Precursor Molecules ◽  
Bovine Neutrophils ◽  
Bovine Neutrophil

Bactenecins are highly cationic polypeptides of bovine neutrophil granules and exert in vitro a potent antimicrobial activity. We have previously purified two bactenecins, designated in an abbreviated form Bac7 and Bac5 from their approximate molecular masses of 7 and 5 kD (Gennaro, R., B. Skerlavaj, and D. Romeo. 1989. Infect. Immun. 57:3142-3146). Here we have studied the biosynthesis, processing, and localization of precursors of Bac7 and Bac5 in bovine bone marrow cells of the myeloid lineage. In vitro translation directed by mRNA isolated from these cells has shown that the primary translation products are preprobactenecins of 23.5 and 21 kD, and are processed to polypeptides of 20 and 15.8 kD, respectively. The 20-kD polypeptide is the granule storage form of Bac7, or proBac7, as also demonstrated by Western blot analysis of lysates of peripheral neutrophils. Between 15 and 50 min from the beginning of its biosynthesis the 15.8-kD polypeptide is converted into the 15-kD granule storage form of Bac5, or proBac5. As shown by immunogold EM, proBac7 and proBac5 are sorted and targeted to the matrix of the so called large granules, which are the predominant organelles in the cytoplasm of bovine neutrophils and are the exclusive store of the nonoxidative antimicrobial system of these cells. Solubilization of granules with Triton X-100 with concomitant unmasking of proteases leads to cleavage of the proforms to Bac7 and Bac5. Experiments performed with protease inhibitors suggest that the proteolytic cleavage is catalyzed in detergent-solubilized neutrophils by neutral serine protease(s), very likely derived from the azurophil granules.

Regulation of gene expression in corn (Zea mays L.) by heat shock. II. In vitro analysis of RNAs from heat-shocked seedlings

1983 ◽  
Vol 61 (6) ◽  
pp. 395-403 ◽  
Author(s):  
Chris L. Baszczynski ◽  
David B. Walden ◽  
Burr G. Atkinson
Keyword(s):  
Heat Shock ◽  
Regulation Of Gene Expression ◽  
In Vitro Translation ◽  
Maize Seedlings ◽  
Mrna Pool ◽  
Molecular Masses ◽  
Vitro Analysis ◽  
Shock Proteins ◽  
In Vitro Analysis

Five-day-old maize seedlings subjected to heat shock exhibit a dramatic enhancement in the synthesis of a small group of polypeptides. Isolation of total RNA from control and heat-shocked maize plumules, fractionation of poly(A)+ mRNA by oligo(dT)-cellulose chromatography, and in vitro translations of the RNAs in both the rabbit reticulocyte and the wheat germ systems indicates that there is remarkable fidelity of the mRNA pool obtained from heat-shocked plumules to reproduce in vitro those same polypeptides whose synthesis is greatly elevated in the intact, heat-shocked plumule. Moreover, these heat-shock polypeptides with molecular masses of 108 000, 89 000, 84 000, 73 000, and 18 000 are translated from polyadenylated mRNAs. The absence of a 76 000 dalton heat-shock polypeptide (HSP) and the presence of fewer isoelectric point variants of the 89 000 and 84 000 dalton HSPs among the in vitro translation products suggests that translational and (or) posttranslational regulatory mechanisms might be operative in determining the final spectrum of the maize heat-shock proteins.

scholarly journals Hydroxylamine Assimilation byRhodobacter capsulatusE1F1

2004 ◽  
Vol 279 (44) ◽  
pp. 45485-45494 ◽  
Author(s):  
Purificación Cabello ◽  
Carmen Pino ◽  
M. Francisca Olmo-Mira ◽  
Francisco Castillo ◽  
M. Dolores Roldán ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Methyl Viologen ◽  
Rhodobacter Capsulatus ◽  
Genomic Library ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Resting Cells

Rhodobacter capsulatusE1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including thehcpgene coding for a hybrid cluster protein (HCP). Expression ofhcpis probably regulated by a nitrite-sensitive repressor encoded by the adjacentnsrRgene. A His6-HCP was overproduced inEscherichia coliand purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammoniain vitrowith methyl viologen as reductant. The apparentKmvalues for NH2OH and methyl viologen were 1 mmand 7 μm, respectively, at the pH and temperature optima (9.3 and 40 °C). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents.R. capsulatusE1F1 grew phototrophically, but not heterotrophically, with 1 mmNH2OH as nitrogen source, and up to 10 mmNH2OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented byl-methionine-d,l-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However,R. capsulatusB10S, a strain lacking the wholehcp-nasregion, did not grow with 1 mmNH2OH. Also,E. colicells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH2OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.

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