scholarly journals Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase

1996 ◽  
Vol 317 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Rathi GANGESWARAN ◽  
Robert R. EADY
Keyword(s):  
Nitrate Reductase ◽  
Electron Donor ◽  
Electrospray Mass Spectrometry ◽  
31P Nmr ◽  
Azotobacter Vinelandii ◽  
Redox Couples ◽  
Related Organism ◽  
Sds Page ◽  
Terminal Amino ◽  
Electron Donation

Flavodoxins synthesized by Azotobacter vinelandii strain UW 136 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter chroococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem. J. 277, 313–319]. Electrospray mass spectrometry gave Mr values for the polypeptides of 19430±3 and 19533±5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (Δ = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at Δ = -142.1 p.p.m. and AvFld 2 at Δ = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone–hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4+-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.

scholarly journals Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor

1993 ◽  
Vol 293 (1) ◽  
pp. 101-107 ◽  
Author(s):  
R N Pau ◽  
M E Eldridge ◽  
D J Lowe ◽  
L A Mitchenall ◽  
R R Eady
Keyword(s):  
Azotobacter Vinelandii ◽  
H2 Evolution ◽  
Titration Curves ◽  
Sds Page ◽  
Metal Requirement ◽  
Alternative Nitrogenase ◽  
Terminal Amino ◽  
Iron Molybdenum Cofactor ◽  
A New Species

Nitrogenase-3 of Azotobacter vinelandii is synthesized under conditions of molybdenum and vanadium deficiency. The minimal metal requirement for its synthesis, and its metal content, indicated that the only transition metal in nitrogenase-3 was iron [Chisnell, Premakumar and Bishop (1988) J. Bacteriol. 170, 27-33; Pau, Mitchenall and Robson (1989) J. Bacteriol. 171, 124-129]. A new species of nitrogenase-3 has been purified from a strain of A. vinelandii (RP306) lacking structural genes for the Mo- and V-nitrogenases and containing a mutation which enables nitrogenase-3 to be synthesized in the presence of molybdenum. SDS/PAGE showed that component 1 contained a 15 kDa polypeptide which N-terminal amino acid sequence determination showed to be encoded by anfG. This confirms that nitrogenase-3, like V-nitrogenase, comprises three subunits. Preparations of the nitrogenase-3 from strain RP306 contained 24 Fe atoms and 1 Mo atom per molecule. Characterization of the cofactor centre of the enzyme by e.p.r. spectroscopy and an enzymic cofactor assay, together with stimulation of the growth of strain RP306 by Mo, showed that nitrogenase-3 can incorporate the Mo-nitrogenase cofactor (FeMoco) to form a functional enzyme. The specific activities (nmol of product produced/min per mg of protein) determined from activity titration curves were: under N2, NH3 formation 110, with concomitant H2 evolution of 220; under argon, H2 evolution 350; under 10% acetylene (C2H2) in argon, ethylene (C2H4) 58, ethane (C2H6) 26, and concomitant H2 evolution 226. The rate of formation of C2H6 was non-linear, and the C2H6/C2H4 ratio strongly dependent on the ratio of nitrogenase components.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

scholarly journals Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

1990 ◽  
Vol 171 (2) ◽  
pp. 565-570 ◽  
Author(s):  
K Ritter ◽  
H Brestrich ◽  
B Nellen ◽  
H Kratzin ◽  
H Eiffert ◽  
...  
Keyword(s):  
Infectious Mononucleosis ◽  
Triosephosphate Isomerase ◽  
Clinical Symptoms ◽  
Glycolytic Enzyme ◽  
Amino Acid Sequences ◽  
Cellular Proteins ◽  
51Cr Release ◽  
Terminal Amino Acid ◽  
Sds Page ◽  
Terminal Amino

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.

scholarly journals PROTEINS OF PAROTOID GLAND SECRETIONS FROM TOADS OF THE GENUS BUFO

2000 ◽  
pp. 1-7 ◽  
Author(s):  
David Perry
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Mass Range ◽  
Relative Molecular Mass ◽  
Terminal Amino Acid ◽  
Banding Patterns ◽  
Freeze Dried ◽  
Sds Page ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Freeze-dried parotoid gland secretions from toads of the genus Bufo contained large proportions of protein (25-35% by weight). SDS-PAGE suggested that secretions from several species of Bufo contained mixtures of proteins in the relative molecular mass range of approximately 12 - 200 kDa, which exhibited markedly different banding patterns from species to species. These proteins were presumably not discovered before because the previous extraction procedures used with these secretions were designed to examine low molecular mass compounds and would denature the proteins. SDS-PAGE of secretions from B. mauritanicus and B. calamita are shown here. The N-terminal amino acid sequence of one of the bands (approx. 58 kDa) of B. mauritanicus was found to be LPIPAFPGLDHGF and of a B. calamita band (30.5 kDa) was VQVFGLQKEA. No significant similarities to these two sequences and to three separate but partial N-terminal sequences obtained from these species were found in genetic databases.

Electron Transport to Assimilatory Nitrate Reductase in A zotobacter vinelandii

1981 ◽  
Vol 36 (11-12) ◽  
pp. 1030-1035 ◽  
Author(s):  
Hermann Bothe ◽  
Klaus-Peter Häger
Keyword(s):  
Electron Transport ◽  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Azotobacter Vinelandii ◽  
Additive Effects ◽  
Electron Carrier ◽  
Transport Chain ◽  
Solubilized Enzyme ◽  
Electron Carriers ◽  
Bound Electron

Abstract Assimilatory nitrate reductase was particle-bound in extracts from Azotobacter vinelandii. Nitrate reduction by particle fractions was dependent on NADPH and a particle-bound electron carrier. When the enzyme was solubilized from the particles by treatment with detergents, the particle-bound electron carrier could be substituted by ferredoxin or flavodoxin. Flavodoxin reduced at the expense of photoreduced deazaflavin was much more efficient than ferredoxin in transferring electrons to nitrate reductase. The addition of both ferredoxin and flavodoxin to the assays with photoreduced deazaflavin gave additive effects. With the solubilized enzyme, NADPH only poorly supported nitrate reduction even after the addition of electron carriers. The experiments indicate that A. vinelandii utilizes an electron transport chain between NADPH and nitrate reductase with some properties similar to those described for the generation of reductants to nitrogenase.

High Expression of Human Amelogenin in E. Coli

1996 ◽  
Vol 10 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D. Deutsch ◽  
E. Chityat ◽  
M. Hekmati ◽  
A. Palmon ◽  
Y. Farkash ◽  
...  
Keyword(s):  
Amino Acid ◽  
Western Blotting ◽  
Rt Pcr ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Expression Plasmid ◽  
Over Expression ◽  
E Coli ◽  
Sds Page ◽  
Terminal Amino

A human cDNA, encoding for the 175-aminoacid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E. coli. The expressed protein was characterized by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing.

scholarly journals Purification and properties of malonyl-CoA synthetase from Rhizobium japonicum

1991 ◽  
Vol 273 (3) ◽  
pp. 511-516 ◽  
Author(s):  
Y S Kim ◽  
H Z Chae
Keyword(s):  
Carbon Sources ◽  
Gel Filtration ◽  
Rhizobium Japonicum ◽  
Purification And Properties ◽  
Malonyl Coa ◽  
Sds Page ◽  
Optimum Ph ◽  
Soybean Nodule ◽  
Terminal Amino ◽  
Single Polypeptide

A novel malonyl-CoA synthetase was found in Rhizobium japonicum bacteroid of the soybean nodule. The levels of the enzyme in the free-living cells grown on a variety of carbon sources including glucose were similar, indicating that this enzyme is not inducible. The malonyl-CoA synthetase from glucose-grown Rhizobium japonicum was purified to homogeneity. The Mr of the enzyme was determined to be 58,000 by gel filtration on a Sephacryl S-300 and by SDS/PAGE respectively, indicating a single polypeptide enzyme. N-Terminal amino acid of the enzyme was methionine but the enzyme preparation contained about 40% de-methionylated protein. The enzyme catalyses the formation of malonyl-CoA, AMP and PPi directly from malonate, CoA and ATP in the presence of Mg2+. High substrate specificity on malonate and ATP was revealed, but Mn2+ could be substituted for Mg2+ without any difference in activity. Optimum pH was 7.9. Kinetic constants, Km and Vmax, for malonate, CoA and ATP were 200 microM and 21.3 mumol/min per mg, 87 microM and 41.7 mumol/min per mg, and 33.3 microM and 29.4 mumol/min per mg respectively. Succinate inhibited the enzyme noncompetitively, whereas AMP and ADP inhibited competitively. Diethylpyrocarbonate and pyridoxal-5′-phosphate severely inhibited the enzyme, but iodoacetamide, p-chloromercuriphenylsulphonate, N-acetylimidazole and phenylglyoxal did not.

The nature of the system (π-C4H7PdCl)2electron donor from 1H, 13C and 31P NMR spectroscopic and electrodialysis studies

1973 ◽  
Vol 54 ◽  
pp. 375-383 ◽  
Author(s):  
V.N. Sokolov ◽  
G.M. Khvostik ◽  
I.Ya. Poddubnyl ◽  
G.P. Kondratenkov ◽  
G.K. Grebenshikov
Keyword(s):  
Electron Donor ◽  
31P Nmr ◽  
13C And 31P Nmr

nasST, two genes involved in the induction of the assimilatory nitrite-nitrate reductase operon (nasAB) of Azotobacter vinelandii

1995 ◽  
Vol 18 (3) ◽  
pp. 579-591 ◽  
Author(s):  
Juan-Carlos Gutierrez ◽  
Francisco Ramos ◽  
Ludwig Ortner ◽  
Maria Tortolero
Keyword(s):  
Nitrate Reductase ◽  
Azotobacter Vinelandii ◽  
Nitrite Nitrate

Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus).

2008 ◽  
Vol 55 (4) ◽  
pp. 753-760 ◽  
Author(s):  
Władysław Polcyn
Keyword(s):  
Mass Spectrometry ◽  
Nitrate Reductase ◽  
Biochemical Properties ◽  
Validation Criteria ◽  
Sds Page ◽  
Mass Spectrometry Identification ◽  
Bradyrhizobium Sp ◽  
Rhizobial Species ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.

Sign in / Sign up

Export Citation Format

Share Document