Isolation of polypeptide chains with heme from the extracellular hemoglobin of Amphitrite ornata (Polychaeta, Annelida)

From the extracellular hemoglobin of Amphitrite ornata four constituent polypeptide chains containing heme and designated AI, AII, BI and BII according to the elution order were obtained by DE52-cellulose ion-exchange chromatography with dithiothreitol (DTT) as a reducing reagent. The NH2-terminal sequences for the chains are AI, Asp-Ser-Asn-Ala; AII, Glu-Tyr-Thr; BI, Asp-Phe-Asn-Thr; and BII, Asp-Ser-Glu. Each of the isolated chains showed spectra similar to those of vertebrate hemoglobins, and they bound oxygen reversibly. Acid urea polyacrylamide gel electrophoresis separated four bands, corresponding to the isolated chain, from the intact extracellular hemoglobin reduced with DTT. These results and our failure to detect an appreciable amount of non-heme protein suggest that the extracellular hemoglobin of A. ornata is composed of four polypeptide chains, each containing a heme.     

Influence of ancillary ligand L in the nitric oxide photorelease by the [Ru(L)(tpy)NO] complex and its vasodilator activity based on visible light irradiation

The photochemical and pharmacological studies of the novel [Ru(L)(tpy)NO]³⁺ L = bpy (2,2′-bipyridine), NH · NHq (quinonediimine) and NH₂.NH₂cat (o-phenylenediamine) were investigated in aqueous medium. The synthesized nitrosyl ruthenium complexes showed nitric oxide (NO) release under light irradiation at 355 nm for [Ru(L)(tpy)NO]³⁺ complex with quantum yield of 0.14 ± 0.02, 0.47 ± 0.03 and 0.46 ± 0.02 mol Einstein⁻¹ for L = bpy, NH · NHq and NH₂ · NH₂cat, respectively, and 0.0065 ± 0.001 mol Einstein⁻¹ for light irradiation at 532 nm for [Ru(NH · NHq)(tpy)NO]³⁺ complex. The photochemical pathway at 355 nm light irradiation was described as a multi-step mechanism, although at 532 nm it was better attributed to a photo-induced electron transfer. The vasorelaxation induced by NO release produced by light irradiation in visible region from physiological solution of [Ru(NH · NHq)(tpy)NO]³⁺ complex was evaluated and compared with sodium nitroprusside (SNP). The results showed very similar vasodilator power between both species.     

Chronic methionine load-induced hyperhomocysteinemia impairs the relaxation induced by bradykinin in the isolated rat carotid

This study investigates the effects of chronic methionine intake on bradykinin (BK)-relaxation. Vascular reactivity experiments were performed on carotid rings from male Wistar rats. Treatment with methionine (0.1, 1 or 2 g kg⁻¹ per day) for 8 and 16 weeks, but not for 2 and 4 weeks, reduced the relaxation induced by BK. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ29548, a selective thromboxane A₂ (TXA₂)/prostaglandin H₂ (PGH₂) receptor antagonist prevented the reduction in BK-relaxation observed in the carotid from methionine-treated rats. Conversely, AH6809, a selective prostaglandin F_2α (PGF_2α) receptor antagonist did not alter BK-relaxation in the carotid from methionine-treated rats. The nitric oxide synthase (NOS) inhibitors L-NAME, L-NNA and 7-nitroindazole reduced the relaxation induced by BK in carotids from control and methionine-treated rats. In summary, we found that chronic methionine intake impairs the endothelium-dependent relaxation induced by BK and this effect is due to an increased production of endothelial vasoconstrictor prostanoids (possibly TXA₂) that counteracts the relaxant action displayed by the peptide.     

Erratum

We studied the structural characteristics of the littoral epilithon in nine lentic water bodies at Hope Bay, Antarctic Peninsula, during summer 2002. At each site we measured the main physical and chemical variables and took epilithic samples for the analysis of dry weigh, ash, ash-free dry weight and chlorophyll a concentration. Distance from the sea of each water body was also considered. One site was selected for sampling lakes and ponds, except for Boeckella lake, where two sampling sites (A and B) were selected due to the heterogeneity of its littoral zone. Three stones for chlorophyll a analysis and another three to estimate dry weight, ash and ash-free dry weight, were taken randomly about 1 m away from the shore-line of each sampling site. Measurements of physical and chemical characteristics were obtained similarly. Water samples for chemical analysis were taken sub-superficially. Lakatoss system of classification and the Autotrophic Index were used to make functional inferences about the epilithic communities. Two PCA analyses were made to classify the water bodies according to environmental features and epilithic variables. In the latter, major patterns in data of epilithon were subsequently interpreted based on environmental data using external validation. Pingüi pond, located in the middle of the penguin rookery, was considered as a passive sample in both PCA analyses due to its extreme characteristics. Limnological features of the studied water bodies were similar to those of other Maritime Antarctic lakes. According to the Lakatoss index, 60% of the sampled lakes had high epilithic mass and a same proportion showed an inorganic type of epilithon The fact that 40% of the water bodies were autotrophic confirmed the importance of benthos as primary producer. According to environmental features, the well-defined groups of lakes emerged from the PCA were mainly determined by distance from the sea, pH, conductivity and salinity, and corresponded to the principal hydrological basins found in the region. Based on the results of the second PCA, littoral epilithon was affected by nitrate concentration and conductivity. In this ordination, water bodies from the same hydrological basin were separated probably as a result of a very complex inter-play of factors with a site-specific response to particular microhabitat characteristics.     

Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase

This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.     

Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18 A resolution: clues for a mechanism for allosteric regulation.

The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine N epsilon atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N delta atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5 degrees with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 degrees rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.     

Effects of ambient water vapor pressure and temperature on evaporative water loss inPeromyscus maniculatus andMus musculus

Summary Oxygen equilibria, ligand-binding kinetics and some other physicochemical properties are reported for erythrocruorins of two intertidal polychaetes:Marphysa sanguinea, which inhabits simple, relatively stagnant burrows, andDiopatra cuprea, which inhabits impermeable, parchment-like tubes that are vigorously ventilated.Marphysa erythrocruorin has a higher O₂ affinity, which is less pH dependent (at pH 7.3 and 20°C, half-saturation O₂ tension,P ₅₀, and Bohr factor, =logP ₅₀/pH, are 0.8 mm Hg and –0.25, respectively) than the corresponding parameters (P ₅₀=5.5; =–0.86) inDiopatra (Figs. 1 and 2). In contrast to vertebrate haemoglobins, inorganic salts increase erythrocruorin O₂ affinity (Fig. 3). The kinetic rates of ligand binding and dissociation ofMarphysa andDiopatra erythrocruorins (Tables 1 and 2) correlate well with the measured O₂ affinities and appear to illustrate basic molecular adaptations of the two species to their respective micro-environmental conditions.