Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.
A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper. (+info)
Negligible amount of copper in hepatic L-tryptophan 2,3-dioxygenase.
During the purification of L-tryptophan 2,3-dioxygenase, a protohemoprotein from rat liver, both copper and heme contents of the preparations were found to be progressively increased as purification proceeded. However, the greater part of copper was removed in the late stages of the purification giving a copper to heme ratio less than 0.4. The small amounts of copper could further be reduced by one-half, by a mild treatment of enzyme with chelators such as ethylenedi aminetetraacetate, without any accompanying decrease in enzymatic activity. Since the turnover number of these enzyme preparations expressed per mol of enzyme-bound heme, 200 to 277 min-1 at 25 degrees, were either comparable to or slightly higher than those reported with homogeneous enzyme preparations, the heme in the preparation was considered to be of fully active L-tryptophan 2,3-dioxygenase and, therefore, such a small ratio of copper to heme, 0.1 to 0.3, indicated that copper is not a constituent of L-tryptophan 2,3-dioxygenase of rat liver. The findings were thus inconsistent with the results of Brady et al. (Brady, F. O., Monaco, M. E. Forman, H. J. Schutz, G., and Feigelson, P. (1972) J. Biol. Chem. 247, 7915-7922), who found that L-tryptophan 2,3-dioxygenase contained 2 g atoms of copper and 2 mol of heme/mol of enzyme. Possible reasons for this discrepancy have been discussed. (+info)
Ruminant placental lactogens act as antagonists to homologous growth hormone receptors and as agonists to human or rabbit growth hormone receptors.
Growth hormone receptor (GHR)-mediated activity of ruminant placental lactogens (PLs) and ovine (o) GH was compared, using cells transfected with full size human (h), rabbit (rb), and oGHRs. All three PLs acted as agonists in heterologous bioassays, whereas in homologous bioassays in cells transfected with oGHRs they antagonized the oGH activity. Despite these differences, oGH and PLs bound with similar affinity to the oGHR extracellular domain (oGHR-ECD), indicating that the binding occurs through hormone site I. Gel filtration of complexes between oPL and oGHR-ECD showed a 1:1 stoichiometry, confirming this conclusion. The oPL T185D and bPL T188D, which exhibited weak biological activity mediated through GHRs, behaved as site I antagonists, whereas oPL G130R and bPL G133R formed a 1:1 complex with GHR-ECDs and bound to h/rb/oGHR-ECDs with affinity similar to that of wild-type oPL. They had no agonistic activity in all models transfected with h/rb and oGHRs, but were antagonistic to all of them. In conclusion, ruminant PLs antagonize the activity of oGH in homologous systems, because they cannot homodimerize oGHRs, whereas in heterologous systems they act as agonists. The structural analysis hints that minor differences in the sequence of the GHR-ECDs may account for this difference. Since the initial step in the activity transduced through cytokine/hemapoietic receptors family is receptor homodimerization or heterodimerization, we suggest that the question of homologous versus heterologous interactions should be reexamined. (+info)
Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase.
Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex. (+info)
Release of copper ions from the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants.
Point mutations of Cu,Zn-superoxide dismutase (SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that the Swedish FALS Cu,Zn-SOD mutant, D90A, exhibited an enhanced hydroxyl radical-generating activity, while its dismutation activity was identical to that of the wild-type enzyme (Kim et al. 1998a; 1998b). Transgenic mice that express a mutant Cu,Zn-SOD, Gly93 --> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the cDNA for the FALS G93A mutant, overexpressed the protein in E. coli cells, purified the protein, and studied its enzymic activities. Our results showed that the G93A, the D90A, and the wild-type enzymes have identical dismutation activity. However, the hydroxyl radical-generating activity of the G93A mutant was enhanced relative to those of the D90A and the wild-type enzyme (wild-type < D90A < G93A). These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules (wild-type < D90A < G93A). The released copper ions can enhance the Fenton-like reaction to produce hydroxyl radicals and play a major role in the oxidative damage of macromolecules. Thus, the FALS symptoms may be associated with the enhancements in both the free radical-generating activity and the releasing of copper ions from the mutant enzyme. (+info)
Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes.
Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation. (+info)
Interference in the quantitation of methylated arsenic species in human urine.
The aim of this paper is to report on the presence of chemical interferences in the quantitation of methylated arsenic species in human urine when using a method based on selective volatile arsine species generation, chromatographic separation, and hydride generation atomic absorption spectrometry (HGAAS) detection. An abnormal profile of methylated arsenic species characterized by the absence of the peak corresponding to dimethylarsinic acid (DMA) was observed in urine from some individuals exposed to arsenic via drinking water and living in rural communities of northwestern Argentina. The absence of this peak persisted even after the addition of known amounts of DMA to the samples. However, the DMA peak appeared after urine digestion with hydrochloric acid (2M). Samples showing interferences were provided by individuals who had mate consumption and coca-leaf chewing habits. Because the relative proportions of methylated arsenic species present in urine have been used to evaluate the efficiency of the methylation process, interferences in the formation or detection of methylarsines may cause underestimation of As exposure and also lead to erroneous conclusions about relative biomethylation efficiencies. Therefore, we recommend that urine samples should be digested with 2M HCl before performing speciation analysis using HGAA techniques. Further studies on the impact of this type of interferences on other arsenic speciation methods are also required. (+info)
Determination of tin, vanadium, iron, and molybdenum in various matrices by atomic absorption spectrometry using a simultaneous liquid-liquid extraction procedure.
An atomic-absorption spectrometric method is described for the determination of tin, vanadium, iron, and molybdenum in two certified reference materials, food samples, and petroleum crude. After treatment with acids, these elements are separated from matrix elements by simultaneous solvent extraction of 5,5'-methylenedisalicylohydroxamic acid complexes from HCl/NaClO4 solution into an isobutyl methyl ketone/tributyl phosphate solution. The detection limits range from 0.018 to 0.19 microg/mL (n = 3), and the relative standard deviations do not exceed 2.0% at levels of 0.5, 0.6, 2.0, and 7.0 microg/mL of Fe, Mo, V, and Sn, respectively. The method is selective and suffers only from interference by Zr(IV), Ti(IV), Th(IV), W(VI), PO4(3-), and F-. (+info)