ABSTRACT
Regulation of antioxidant enzymes is critical to control the levels of reactive oxygen species in cell compartments highly susceptible to oxidative stress. In this work, we studied the regulation of a chloroplastic iron superoxide dismutase (Fe-SOD) from Lingulodinium polyedrum (formerly Gonyaulax polyedra) under different physiological conditions. A cDNA-encoding Fe-SOD was isolated from this dinoflagellate, showing high sequence similarity to cyanobacterial, algal, and plant Fe-SODs. Under standard growth conditions, on a 12:12-h light-dark cycle, Lingulodinium polyedrum Fe-SOD exhibited a daily rhythm of activity and cellular abundance with the maximum occurring during the middle of the light phase. Northern analyses showed that this rhythmicity is not related to changes in Fe-SOD mRNA levels, indicative of translational regulation. By contrast, conditions of metal-induced oxidative stress resulted in higher levels of Fe-SOD transcripts, suggesting that transcriptional control is responsible for increased protein and activity levels. Daily (circadian) and metal-induced up-regulation of Fe-SOD expression in L. polyedrum are thus mediated by different regulatory pathways, allowing biochemically distinct changes appropriate to oxidative challenges.
Subject(s)
Superoxide Dismutase/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , DNA Primers , DNA, Complementary , Dinoflagellida , Metals , Molecular Sequence Data , Oxidative Stress , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Superoxide Dismutase/chemistry , Superoxide Dismutase/geneticsABSTRACT
Alignment of the primary structures of the hydrozoan photoproteins, aequorin, mitrocomin, clytin and obelin showed very strong amino acid sequence identities. The Ca(2+)-binding sites of the proteins were found to be highly conserved. The Ca(2+)-binding sites were also homologous to the Ca(2+)-binding sites of other Ca(2+)-binding proteins. However, aequorin, mitrocomin, clytin and obelin differed from other Ca(2+)-binding proteins in that they contained a relatively large number of cysteine, tryptophan, histidine, proline and tyrosine residues, suggesting that these residues may have evolved as part of the light-emitting mechanism. Construction of a phylogenetic tree showed that aequorin, mitrocomin, clytin and obelin form a closely related group of proteins.
Subject(s)
Biological Evolution , Calcium-Binding Proteins/chemistry , Luminescent Proteins/chemistry , Porifera/metabolism , Aequorin/chemistry , Amino Acid Sequence , Animals , Binding Sites , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Databases, Factual , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , Porifera/genetics , Sequence Homology, Amino AcidABSTRACT
The primary structure of mitrocomin consists of 190 amino acid residues, with three Ca(2+)-binding sites and a tyrosine residue at the C-terminus. Mitrocomin shows an amino acid sequence homology of 67.9% and 60.7% when compared with aequorin and clytin, respectively. The amino acid residues Cys152, His58, His169, Trp12, Trp86, Trp108, Trp129 and Trp173 are conserved in all three photoproteins, suggesting that they play a role in light emission.
Subject(s)
Calcium-Binding Proteins/biosynthesis , DNA, Complementary/chemistry , Luminescent Proteins/biosynthesis , Scyphozoa/metabolism , Aequorin/chemistry , Amino Acid Sequence , Animals , Base Sequence , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Cloning, Molecular/methods , Consensus Sequence , DNA, Complementary/genetics , Luminescence , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Restriction Mapping , Scyphozoa/genetics , Sequence Homology, Amino AcidABSTRACT
Tryptic digests of purified recombinant apoaequorin were analyzed, before and after reduction with DTT, by fast atom bombardment mass spectrometry. The results showed that apoaequorin contains a disulfide bond between Cys145 and Cys152 and that the reduction of this bond is involved in the regeneration of aequorin.
Subject(s)
Aequorin/chemistry , Disulfides/analysis , Protein Structure, Secondary , Aequorin/metabolism , Amino Acid Sequence , Animals , Dithiothreitol/pharmacology , Escherichia coli/metabolism , Molecular Sequence Data , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Scyphozoa , Spectrometry, Mass, Fast Atom Bombardment/methods , TrypsinABSTRACT
The H2-oxidation, H2-production and H-3H-exchange activities of the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) were almost completely abolished by Hg(II) and the organic mercurials p-chloromercuribenzoate (pCMB) and p-hydroxymercuriphenylsulphonate. The thiol-modifying reagents N-ethylmaleimide, iodoacetate, dithionitrobenzoate and 2-nitro-5-thiocyanobenzoate had no effect on the activities. Kinetic and spectroscopic measurements suggest that inactivation by pCMB involves at least two reactions; a rapid reaction that is reversed by thiols, and a second, slower and irreversible reaction that occurs at high concentrations of the mercurial. The irreversible reaction was associated with loss of visible absorbance, indicative of a disrupted iron sulphur cluster(s). The effects on the H-3H-exchange activity indicate that the reversible modification affects the H2-activating site. Enzyme that had lost activity due to pCMB treatment, or during long-term storage, was reactivated by thiols. This reactivation was followed by a slower irreversible inactivation, as also occurred with native enzyme; the inactivation was O2 dependent and it was partly prevented by catalase, suggesting that H2O2 may be involved.
