Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.
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PMID:Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme. 359

The isolation and purification of a protein which is the presumed product of the chlA gene has been achieved. This protein, which we have named Protein PA, has been isolated from the soluble fraction of a chlB mutant. The protein was identified by its ability to activate nitrate reductase (EC.1.7.99.4) when mixed with a soluble fraction derived from a chlA mutant. The protein has a molecular weight of about 72 000 and is composed of a single polypeptide chain. Antiserum specific for Protein PA has been produced. Removal of Protein PA from the soluble fraction of chlB mutant by immunoprecipitation with this antiserum leads to the loss of the ability of the preparation to activate nitrate reductase when mixed with a soluble fraction from a chlA mutant. Protein PA, therefore, performs an essential but as yet undefined role in the activation process. Employing this antiserum Protein PA could be quantified by rocket immunoelectrophoretic analysis. The activity of the isolated Protein PA is low, since comparatively large amounts of Protein PA are required to activate the nitrate reductase present in the soluble fraction of the chlA mutant. The mixing of Protein PA with the chlA mutant soluble fraction leads to activation of nitrate reductase in both a soluble and a membranous form, as is the case when the complete soluble fraction of the chlB mutant is used in place of Protein PA. After activation, however, only a small proportion (15%) of the Protein PA is associated with the newly formed membranous material.
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PMID:Identification and purification of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K12. 631 66

An enzymatic activity which modifies nitrate reductase has been identified in the cytoplasmic membrane of Escherichia coli. This activity changes subunit B to a form with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels (B'). The B' polypeptide produced by this modifying enzyme was compared to an apparently identical polypeptide identified in the precursor form of nitrate reductase which can be found in the cytoplasm of all strains and in the membrane of mutants defective in the insertion of nitrate reductase. These B' polypeptides were all identical with respect to mobility on gradient sodium dodecyl sulfate gels and peptides produced by limited digests using trypsin, papain, and Staphylococcus aureus V8 protease. When compared to subunit B, the proteolytic gel maps of B' polypeptides showed minor differences. From the identity of the modified B' with precursor B', the ability to convert B into B' in vitro and the in vivo nature of B' as a precursor of B, it was concluded that the modification of B to B' is a reversible process and is due to the removal of one or more small nonprotein molecules.
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PMID:An activity from Escherichia coli membranes responsible for the modification of nitrate reductase to its precursor form. 634 96

The role of the cnxH+ gene specified polypeptide in the formation and function of the NADPH-nitrate reductase in Aspergillus nidulans was examined with the use of two complementing mutant strains which were grown as forced heterocaryons in the presence of nitrate. The niaD-421 structural gene mutant and the cnxH-318 co-factor gene mutant produce two components of the NADPH-cytochrome c reductase co-activity which can be distinguished by their enzymatic and physical behavior. This combination enabled us to isolate the de novo synthesis of niaD+ gene specified protomers from the constitutively formed co-factor at two stages of development. The proportion of induced and constitutively formed protomers in the isolated holoenzyme was measured after pulsing with [3H]-histidine or [14C]-histidine prior to induction with nitrate. The newly formed nitrate reductase was resolved by agarose gel electrofocusing and activity staining. In vivo assembly of a 7.8s enzyme in the heterocaryotic mycelium of the above strains is apparently achieved by the convener action of the cnxH+ gene directed polypeptide from the niaD- strain on the niaD+ gene directed protomers of the cnxH- partner. This occurs with or without Mo as a co-factor.
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PMID:A convener role for the cnxH gene specified component in the NADPH-nitrate reductase fron Aspergillus nidulans. 642 61

The synthesis of nitrate reductase by a parental Escherichia coli K12 strain and its isogenic chlA and chlB mutants has been analyzed by protein double labelling with L-[4,5-3H]leucine and sulphur-35 and by immunoprecipitation using specific antiserum. The chlA and chlB mutants although defective in nitrate reductase activity retain the ability to synthesise the different polypeptides that are normally required for functional enzyme activity. In addition the data shows the following. 1. These polypeptides are present in unequal quantities in the membrane and in the cytoplasm of the cells. The chlB mutant synthesizes three times more nitrate reductase than the chlA mutant. 2. The subunit composition of the membrane-bound nitrate reductase present in the two mutants is different. 3. Membrane preparations from the chlB mutant contain the three subunits alpha, beta, gamma in a ratio which is similar to the wild type. 4. In the chlA mutant the two subunits beta and gamma are missing and the level of alpha subunit is very low. In the same membrane a 48,000-Mr subunit (polypeptide beta') precipitable by nitrate reductase antiserum has been found. The chlA and chlB mutants accumulate the three subunits alpha, beta and gamma in different proportion and concentrations in the cytoplasm unlike the parental strain. 5. The cytoplasm from the chlA mutant also contains the beta' polypeptide found in the membrane fraction of this mutant and in addition contain another polypeptide designated alpha' of molecular weight 105,000 which is precipitated by the nitrate reductase antiserum. The formation of particulate active nitrate reductase can be achieved by mixing the supernatant fractions of the chlA and chlB mutants (complementation) and procedes by two distinct but mutually dependent stages. Following reconstitution of activity the two peptides alpha' and beta' present in the supernatant fraction of the chlA mutant, disappear. Analysis of the immunoprecipitate polypeptides present in both the soluble and particulate nitrate reductase protein after reconstitution suggests that these polypeptides are precursors of the alpha and beta subunits following a process that remains to be elucidated.
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PMID:Precursor forms of the subunits of nitrate reductase in chlA and chlB mutants of Escherichia coli K12. 699 Dec 54

Insertion of nitrate reductase into the Escherichia coli cytoplasmic membrane was examined by following the fate of pulse-labeled enzyme in both the membrane and cytoplasm during various times after the addition of an unlabeled chase. The polypeptide composition of this labeled enzyme was determined by autoradiography of immunoprecipitated material after separation on sodium dodecyl sulfate-polyacrylamide gels. The data presented here indicate that immediately after appropriate insertion of the enzyme into the membrane, a post-translational event occurs which converts the cytoplasmically synthesized form of subunit B (B') to the form found in the completely assembled enzyme (B). B' is distinguished from B by its more rapid electrophoretic mobility. B' was found in the cytoplasm of all strains tested, in the membrane of strains with defects in enzyme insertion (hemA and chlE), and as a transient component in the membrane of wild-type cells.
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PMID:New mechanism for post-translational processing during assembly of a cytoplasmic membrane protein? 702 18

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide Mr = 150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrance protein Mr = 150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are co-regulated and that the active enzyme has a role in regulating its own synthesis.
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PMID:Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans. 719 81

Specific antiserum to the membrane nitrate reductase of Staphylococcus aureus was derived from immunoprecipitates on crossed immunoelectrophoresis plates. Analysis of the cytoplasmic and membrane forms of the enzyme in cells grown with nitrate and azide indicated their identity, and in each case, the major subunit, Mr 140,000, was converted by trypsin to a polypeptide, Mr 112,000, without loss of enzyme activity or immunological reactivity.
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PMID:Membrane and cytoplasmic nitrate reductase of Staphylococcus aureus and application of crossed immunoelectrophoresis. 719 72

The mob mutants in Escherichia coli are pleiotropically defective in all molybdoenzyme activities. They synthesise molybdopterin, the unique core of the molybdenum cofactor, but are unable to attach the GMP moiety to molybdopterin to form molybdopterin guanine dinucleotide, the functional molybdenum cofactor in Escherichia coli. A partially purified preparation termed protein FA (protein factor d'association), is able to restore molybdoenzyme activities to broken cell preparations of mob mutants. A fragment of DNA capable of complementing mob mutants has been isolated from an E. coli genomic library. Strains carrying this DNA in a multicopy plasmid, express 30-fold more protein FA activity than the wild-type bacterium. Protein FA has been purified to homogeneity by a combination of ion-exchange, affinity and gel-filtration chromatography. Protein FA consists of a single polypeptide of molecular mass 22 kDa and is monomeric in solution. N-terminal amino acid sequencing confirmed that protein FA is a product of the first gene at the mob locus. The purified protein FA was required in stoichiometric rather than catalytic amounts in the process that leads to the activation of the precursor of the molybdoenzyme nitrate reductase, which is consistent with the requirement of a further component in the activation.
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PMID:Isolation of protein FA, a product of the mob locus required for molybdenum cofactor biosynthesis in Escherichia coli. 802 May 7

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.
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PMID:Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha. 811 78


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