Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.     

Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. RESULTS: Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB. CONCLUSIONS: Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.     

Expression of the gene for the alpha-subunit of inhibin in human adrenocortical tumours

BACKGROUND: To determine whether the pathogenesis of human adrenocortical tumours is associated with variations of inhibin expression, we assayed the mRNA of the alpha-subunit of inhibin in 5 normal adrenals and 48 adrenocortical tumours, including 10 paediatric tumours. RESULTS: mRNA of alpha-subunit of inhibin was detected in all adrenocortical tissues. It was similarly abundant in the three pathological groups of adult tumours (benign, suspect and malignant) and in normal adrenal tissues, irrespective of the hormonal pattern. However, in paediatric tumours, the levels of the mRNA for the alpha-subunit of inhibin were significantly higher than those in adult tumours (p < 0.01). CONCLUSION: Inhibin is more abundant in the foetal than in the adult adrenal cortex and therefore these data suggest that the paediatric tumours may have a foetal pattern.     

Multicopy crystallographic refinement of a relaxed glutamine synthetase from Mycobacterium tuberculosis highlights flexible loops in the enzymatic mechanism and its regulation

The crystal structure of glutamine synthetase (GS) from Mycobacterium tuberculosis determined at 2.4 A resolution reveals citrate and AMP bound in the active site. The structure was refined with strict 24-fold noncrystallographic symmetry (NCS) constraints and has an R-factor of 22.7% and an R-free of 25.5%. Multicopy refinement using 10 atomic models and strict 24-fold NCS constraints further reduced the R-factor to 20.4% and the R-free to 23.2%. The multicopy model demonstrates the range of atomic displacements of catalytic and regulatory loops in glutamine synthesis, simulating loop motions. A comparison with loop positions in substrate complexes of GS from Salmonella typhimurium shows that the Asp50 and Glu327 loops close over the active site during catalysis. These loop closures are preceded by a conformational change of the Glu209 beta-strand upon metal ion or ATP binding that converts the enzyme from a relaxed to a taut state. We propose a model of the GS regulatory mechanism based on the loop motions in which adenylylation of the Tyr397 loop reverses the effect of metal ion binding, and regulates intermediate formation by preventing closure of the Glu327 loop.     

High pressure, a tool for exploring heme protein active sites

High pressure is an interesting and suitable parameter in the study of the dynamics and stability of proteins. The effects of pressure on proteins delineates its volumic (deltaV degrees ) and energetic (deltaG degrees ) parameters. An enormous amount of effort has been invested by several laboratories in developing basic theory and high pressure techniques that allow the determination of barotropic parameters. Cytochrome P450s, one of the largest super families of heme proteins, are good models for high pressure studies. Two distinct pressure-induced spin transitions of the heme iron in the active site and a P450 to P420 inactivation process have been characterized. The obtained reaction volumes of these two processes for a series of analog-bound cytochrome P450s are compared. We have shown that both the spin volume and the inactivation volume are dependent on the substrate analogs which are known to modulate the polarity and hydration of the heme pocket. Several linear correlations were found between these reaction volumes and the physico-chemical properties of the heme protein such as the polarity-induced exposure of tyrosines, the hydration of the cytochrome CYP101 heme pocket, and the mobility and binding of the substrates indicate that they constitute the main contribution to the complex thermodynamic reaction volume parameters. This interpretation allows us to conclude that cytochrome CYP101, CYP2B4 and CYP102 possess a similar mechanism of substrate binding. Interestingly the barotropic behaviors of monomeric cytochrome P450s are quite different from those of oligomeric and hetorooligomeric cytochrome P450s. The interactions of heterooligomeric subunits influence the stability of individual cytochrome P450s and the asymmetric organization of subunits which can control and modulate the activity and the recognition with NADPH-cytochrome P450 reductase.     

Changes in mitochondrial electron partitioning in response to herbicides inhibiting branched-chain amino acid biosynthesis in soybean

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.     

Nontyphoidal salmonellae in United Kingdom badgers: prevalence and spatial distribution

Eighteen (72%) of 25 badger social groups were found to excrete Salmonella enterica serovar Ried, S. enterica serovar Binza, S. enterica serovar Agama, or S. enterica serovar Lomita. Each serovar was susceptible to a panel of antimicrobials. Based on results of pulsed-field gel electrophoresis, the S. enterica serovar Agama and S. enterica serovar Binza isolates were very similar, but two clones each of S. enterica serovar Lomita and S. enterica serovar Ried were found. Badgers excreting S. enterica serovar Agama were spatially clustered.     

S-nitrosylation signaling in cell biology

S-Nitrosylated proteins form when a cysteine thiol reacts with nitric oxide (NO) in the presence of an electron acceptor to form an S-NO bond. Under physiological conditions, this posttranslational modification affects the function a wide array of cell proteins, ranging from ion channels to nuclear regulatory proteins. Recent evidence suggests that 1) S-nitrosylated proteins can be synthesized by exposure of specific redox-active motifs to NO, through transnitrosation/transfer reactions, or through metalloprotein-catalyzed reactions; 2) S-nitrosothiols can be sequestered in membranes, lipophilic protein folds, or in vesicles to preserve their activity; and 3) S-nitrosothiols can be degraded by a number of enzymes systems. These recent insights regarding the bioactivities, molecular signaling pathways, and metabolism of endogenous S-nitrosothiols have suggested several new therapies for disease ranging from cystic fibrosis to pulmonary hypertension.     

Areas, cradles and museums: the latitudinal gradient in species richness

Although numerous factors are postulated to be responsible for the gradient of increasing taxon richness towards lower latitudes, it has recently been suggested that the primary determinant is geographic area. This area model is appealing in its logic, but there is little empirical evidence to support it and several other mechanisms might also interact to obscure its effects. Nonetheless, the model has highlighted several fundamental issues concerning range size, speciation and extinction that, despite their considerable significance, remain poorly understood.