Effect of denaturants on the structural properties of soybean lipoxygenase-1.

The effect of chemical (urea) and physical (temperature and high pressure) denaturation on the structural properties of soybean lipoxygenase-1 (LOX1) was analyzed through dynamic fluorescence spectroscopy and circular dichroism. We show that the fluorescence decay of the native protein could be fitted by two lorentzian distributions of lifetimes, centered at 1 and 4 ns. The analysis of the urea-denatured protein suggested that the shorter distribution is mostly due to the tryptophan residues located in the N-terminal domain of LOX1. We also show that a pressure of 2400 bar and a temperature of 55 degrees C brought LOX-1 to a state similar to a recently described stable intermediate "I." Analysis of circular dichroism spectra indicated a substantial decrease of alpha-helix compared with beta-structure under denaturing conditions, suggesting a higher stability of the N-terminal compared with the C-terminal domain in the denaturation process.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

In vivo and in vitro development of mouse pancreatic β-cells in organotypic slices

Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing -cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single -cells. The insulin signal in the embryonic -cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in -cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of -cells.This work was supported by the European Commission (grant QLG1-CT-2001-02233 to TMR, AR and MR), the DFG Research Center for Molecular Physiology of the Brain (CMPB) and the Max-Planck Society (MR)     

Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.     

Ceramide Induces Release of Pro-Apoptotic Proteins from Mitochondria by Either a Ca2+-Dependent or a Ca2+-Independent Mechanism

Several observations have been reported in the last years indicating that ceramide may activate the mitochondrial route of apoptosis. We show here that on addition of either C2- or C16-ceramide to mitochondria isolated from rat heart and suspended in a saline medium, release of cytochrome c and apoptosis-inducing factor (AIF) from the intermembrane space takes place. The release process is Ca2+ -independent and is not inhibited by Cyclosporin A (CsA). For the protein release process to occur, the presence of an oxidizable substrate is required. When mitochondria are suspended in sucrose instead of potassium medium, only short chain C2-ceramide causes cytochrome c release through a Ca2+ -dependent and CsA sensitive mitochondrial permeability transition (MPT) mechanism. The latter effect appears to be related to the membrane potential dissipating ability exhibited by short chain C2-ceramide.     

Functional characterization of an insulin-responsive glucose transporter (GLUT4) from fish adipose tissue

Glucose transport across the plasma membrane is mediated by a family of glucose transporter proteins (GLUTs), several of which have been identified in mammalian, avian, and, more recently, in fish species. Here, we report on the cloning of a salmon GLUT from adipose tissue with a high sequence homology to mammalian GLUT4 that has been named okGLUT4. Kinetic analysis of glucose transport following expression in Xenopus laevis oocytes demonstrated a 7.6 +/- 1.4 mM K(m) for 2-deoxyglucose (2-DG) transport measured under zero-trans conditions and 14.4 +/- 1.5 mM by equilibrium exchange of 3-O-methylglucose. Transport of 2-DG by okGLUT4-injected oocytes was stereospecific and was competed by D-glucose, D-mannose, and, to a lesser extent, D-galactose and D-fructose. In addition, 2-DG uptake was inhibited by cytochalasin B and ethylidene glucose. Moreover, insulin stimulated glucose uptake in Xenopus oocytes expressing okGLUT4 and in isolated trout adipocytes, which contain the native form of okGLUT4. Despite differences in protein motifs important for insulin-stimulated translocation of mammalian GLUT4, okGLUT4 was able to translocate to the plasma membrane from intracellular localization sites in response to insulin when expressed in 3T3-L1 adipocytes. These data demonstrate that okGLUT4 is a structural and functional fish homolog of mammalian GLUT4 but with a lower affinity for glucose, which could in part explain the lower ability of fish to clear a glucose load.     

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.