Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.     

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli

Spheroplasts from aerobically grown wild-type Paracoccus denitrificans cells respire with succinate despite specific inhibition of the cytochrome bc1 complex by myxothiazol. Coupled to this activity, which involves only b-type cytochromes, there is translocation of 1.5-1.9 h+/e- across the cytoplasmic membrane. Similar H+ translocation ratios are observed during oxidation of ubiquinol in spheroplasts from aerobically grown mutants of Paracoccus lacking cytochrome c oxidase, or deficient in cytochrome c, as well as in a strain of E. coli from which cytochrome d was deleted. These observations show that the cytochrome o complex is a proton pump much like cytochrome aa3 to which it is structurally related.     

Strong dependence between phytoplankton and water chemistry in a large temperate lake: spatial and temporal perspective

In lakes, spatial and temporal variability of water chemistry and phytoplankton are characteristic phenomena although often difficult to link together. This motivated us to study their interplay in Lake Vanajanselkä, a eutrophic lake in Finland. We hypothesized that in summer spatial and temporal differences in phytoplankton and water chemistry can be extended in comparison to spring and autumn. Therefore, chlorophyll a and water chemistry was examined by six sampling campaigns with 15 sampling sites over the lake in May–October 2009–2010. In summer, chlorophyll, pH, and oxygen were horizontally and vertically unevenly distributed in the lake, and in the epilimnion pH and oxygen showed a distinct diurnal variability suggesting high photosynthesis during the day. Daily >1 pH unit difference between the sites and 2.5 pH unit difference between the epi- and hypolimnion were found. In agreement with pH and oxygen, NO₃-N and NH₄-N could be unevenly distributed in the epilimnion. In autumn no spatial differences were found, however. The results emphasized that algae and cyanobacteria were responsible, at least partly, for the variability in water chemistry in the surface layer, and short- and long-term gradients in space and time need to be considered when productive lakes are studied.     

ATR-FTIR spectroscopy and isotope labeling of the PM intermediate of Paracoccus denitrificans cytochrome c oxidase

The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes. In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site. In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments. In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine. In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected. Both isotope substitutions had large effects on P(M) minus oxidized difference spectra. A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling. Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra. These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B). Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate. Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M).     

Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) regulates intracellular Ca2+ by mobilizing Ca2+ from a non-mitochondrial store. We have investigated the effects of Ca2+ on the binding of [32P]Ins (1,4,5)P3 to permeabilized rat hepatocytes and a liver plasma membrane-enriched fraction. Increasing the free Ca2+ concentration in the medium from 0.1 nM to 0.7 microM increased the capacity of a high affinity binding component (KD = 2-3 nM) in permeabilized cells by a factor of 10. If the membrane fraction was preincubated at 37 degrees C before binding was measured at 4 degrees C, all of the Ins(1,4,5)P3 receptors were transformed to a low affinity state (KD = 65 +/- 12 nM, Bmax = 3.1 +/- 0.1 fmol/mg, n = 4). When 0.7 microM of Ca2+ was added, the receptors were totally transformed to a high affinity state (KD = 2.8 +/- 0.4 nM, Bmax = 2.7 +/- 0.4 fmol/mg, n = 4). The EC50 of the Ca2(+)-induced interconversion of the Ins(1,4,5)P3 receptor was 140 nM. This Ca2(+)-induced transformation of the Ins(1,4,5)P3 receptor from a low affinity to a high affinity state was associated with an inhibition of the Ins(1,4,5)P3-induced Ca2+ release in permeabilized hepatocytes. These data suggest that the Ins(1,4,5)P3-dependent hormones, by increasing the intracellular Ca2+ concentration, induce a reversible transformation of the receptor from its low affinity state, coupled to the Ca2+ release, to a desensitized high affinity state. Transformation of the receptor may play a role in the oscillatory release of Ca2+ observed in single isolated hepatocytes.     

The calcium binding site in cytochrome aa3 from Paracoccus denitrificans.

A shift in the spectrum of heme a induced by calcium or proton binding, or by the proton electrochemical gradient, has been attributed to interaction of Ca2+ or H+ with the vicinity of the heme propionates in mitochondrial cytochrome c oxidase, and proposed to be associated with the exit path of proton translocation. However, this shift is absent in cytochrome c oxidases from yeast and bacteria [Kirichenko et al. (1998) FEBS Lett. 423, 329-333]. Here we report that mutations of Glu56 or Gln63 in a newly described Ca2+/Na+ binding site in subunit I of cytochrome c oxidase from Paracoccus denitrificans [Ostermeier et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10547-10553] establish the Ca2+-dependent spectral shift in heme a. This shift is counteracted by low pH and by sodium ions, as was described for mammalian cytochrome c oxidase, but in the mutant Paracoccus enzymes Na+ is also able to shift the heme a spectrum, albeit to a smaller extent. We conclude that the Ca2+-induced shift in both Paracoccus and mitochondrial cytochrome aa3 is due to binding of the cation to the new metal binding site. Comparison of the structures of this site in the two types of enzyme allows rationalization of their different reactivity with cations. Structural analysis and data from site-directed mutagenesis experiments suggest mechanisms by which the cation binding may influence the heme spectrum.     

Reappraisal of early Miocene rails (Aves,Rallidae) from central France: diversity and character evolution

In Europe, Miocene rails (Aves, Rallidae) are quite abundant, but their phylogenetic placement in the context of recent forms has remained elusive. Rails from the early Miocene of the Saint‐Gérand‐le‐Puy area in central France were first described in the 19th century, and currently, only two species are recognized, namely Palaeoaramides christyi and Paraortygometra porzanoides. Our examination of the material however suggests the presence of four, likely coeval, species of rail from these deposits. Palaeoaramides eximius, previously synonymized with Palaeoaramides christyi, is here shown to probably be a distinct species, and a previously unrecognized rail, Baselrallus intermedius gen. et sp. nov., is described. To find out how these fossil rails are related to modern Rallidae, we compared them with an extensive sample of extant rails and identified plesiomorphic and derived features for crown group Rallidae. Our assessment does not support a particularly close relationship of either Palaeoaramides to Aramides or Paraortygometra to Crex (Ortygometra), and overall, these fossil rails are more primitive than previously assumed. Based on our observations of the morphology of the previously undescribed humerus of Palaeoaramides, we show this taxon to be outside crown group Rallidae, and perhaps closely related to the early Oligocene taxon Belgirallus. On the other hand, Paraortygometra porzanoides bears a resemblance to recent flufftails (Sarothrura spp.) in some elements, but whether it can be included in a clade together with flufftails is uncertain.