Post-translational Transformation of Methionine to Aspartate Is Catalyzed by Heme Iron and Driven by Peroxide: A NOVEL SUBUNIT-SPECIFIC MECHANISM IN HEMOGLOBIN*

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H₂O₂). Using H₂¹⁸O₂, we verified incorporation of ¹⁸O into the Asp carboxyl side chain confirming the role of H₂O₂ in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.     

Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells

Although it is understood that hydrogen peroxide (H₂O₂) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H₂O₂ in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H₂O₂. The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0  ±  4.7 h versus 28.6  ±  0.8 h, p < 0.05), consistent with a diminished growth rate and H₂O₂ release. Incubation with aminotriazole, a catalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G₀/G₁ phase in hCatTg MAECs compared to the wild-type cells (25.0  ±  0.9 h versus 15.9  ±  1.4 h, p  <  0.05). The hCatTg MAECs also exhibited decreased activities of the cyclin-dependent kinase (Cdk) complexes responsible for G₀/G₁- to S-phase transition in the cell cycle, including the cyclin D–Cdk4 and cyclin E–Cdk2 complexes. Moreover, the reduction in cyclin–Cdk activities in hCatTg MAECs was accompanied by increased protein levels of two Cdk inhibitors, p21 and p27, which inhibit the Cdk activity required for the G₀/G₁- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H₂O₂ scavenger, suggest that endogenously produced H₂O₂ mediates MAEC proliferation by fostering the transition from G₀/G₁ to S phase.     

Influence of diets supplemented with vitamins C and E on pirarucu (Arapaima gigas) blood parameters

This study evaluated the influence of diets supplemented with 500, 800, 1200 mg kg^− 1 of vitamin C (ascorbic acid or AA) and vitamin E (α-tocopherol or α-T) on the physiological responses of pirarucu fed for 2 months. Weight and mortality were not affected by dietary vitamin type or their concentrations. Significant increase (p < 0.05) on the red blood cells count was obtained on treatments with 800 and 1200 mg AA kg^− 1 and on the hemoglobin concentration on treatment with 500 mg α-T kg^− 1 relatively to control. Mean corpuscular volume presented a significant decrease (p < 0.05) on treatment with 800 and 1200 mg AA kg⁻¹ when compared to control. Mean corpuscular hemoglobin concentration was significantly high (p < 0.05) on treatment with 500 mg α-T kg^− 1. Only in vitamin C treatments, we noticed a significant increase (p < 0.05) in the number of leucocytes relative to control. All fish in the vitamin-supplemented treatments, except 500 mg AA kg^− 1, had high total protein values compared to control. Fish treated with 800 or 1200 mg α-T kg^− 1 also showed increases in plasma glucose concentrations. Our results suggest that 800 and 1200 mg AA kg^− 1 are probably the most suitable concentrations for pirarucu diets, although high vitamin E diets are not necessary for quantitative leucocyte increases for this species.     

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer

Human hemoglobin (Hb), which is an α₂β₂ tetramer and binds four O₂ molecules, changes its O₂-affinity from low to high as an increase of bound O₂, that is characterized by ‘cooperativity’. This property is indispensable for its function of O₂ transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O₂-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O₂-binding with no cooperativity, indicating the coexistence of two independent hemes with different O₂-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O₂-binding curve with no cooperativity. Resonance Raman, ¹H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O₂-binding to rHb(αH87G), but it did partially occur with O₂-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O₂-binding, but its absence in the β subunit simply enhances the O₂-affinity of α subunit.     

Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard,bicarbonate-rich freshwater

Summary Carbonic anhydrase (CA) activities in gills and venous blood, acid-base balance, and haematological variables were studied during environmental hypercapnia in rainbow trout (Salmo gairdneri). Batches of 8–10 fish were exposed to about 3 or 13 mmHg in flow-through tests of various duration from 4 h to 80 days.After initial acidosis, blood pH rose above pre-experimental values. At 3 mmHg it became normal again within 21 days, while at 13 mmHg the overshoot lasted for 80 days. In fish acclimated for 3 weeks or more to 13 mmHg , blood HCO ₃ ^– increased four to five times while plasma Cl^– levels were lower and K⁺ higher. Na⁺ levels did not show any consistent trend associated with exposure to hypercapnia. After an initial acidaemia, Hct, Hb, and RBC remained relatively constant.Patterns of change in CA activity differed between gills and erythrocytes. Initially, blood CA decreased at both levels. It then began rising after about 3 weeks and tended to reach pre-experimental values by 80 day's hypercapnia. At 13 mmHg , gill CA increased to twice the pre-experimental level. Compared with blood CA, gill CA appeared to be more specifically involved in fish acclimation to hypercapnia, which demands an increase in blood bicarbonate to provide a sufficient buffering capacity. Increased CA indicates that the gill enzyme may play a more important role than blood CA in acid-base regulation in fish during hypercapnia.Abbreviations CA carbonic anhydrase - Hb haemoglobin - Hct haematocrit value - RBC red blood cells     

Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.     

Kinetics of the acclimational responses of tench to combined hypoxia and hypercapnia

Summary Exposing tench to environmental hypoxia-hypercapnia reduces routine O₂ consumption, sharply decreases arterial O₂ tension and the difference between the water and the blood, and results in marked swelling of the erythrocytes. These changes are rapidly reversed upon return to normoxia.Hypoxic-hypercapnic conditions lower the blood NTP/Hb ratio to a new steady state level within 24 h, by reducing GTP/Hb but not ATP/Hb. A similar selective reduction of eryhtrocytic GTP content forms the initial response of blood incubated in vitro to anoxic conditions.The swelling as well as the reduced GTP/Hb ratio in the erythrocytes appear to improve O₂ loading in the gills during environmental hypoxia-hypercapnia.Symbols and abbreviations a arterial - GTP guanosine triphosphate - Hct hematocrit - I inspired - NTP nucleoside triphosphate - w water     

Partial characterization of a malonyl-CoA-sensitive carnitine O-palmitoyltransferase I from Macrobrachium borellii (Crustacea: Palaemonidae)

The shuttle system that mediates the transport of fatty acids across the mitochondrial membrane in invertebrates has received little attention. Carnitine O-palmitoyltransferase I (EC 2.3.1.21; CPT I) is a key component of this system that in vertebrates controls long-chain fatty acid β-oxidation. To gain knowledge on the acyltransferases in aquatic arthropods, physical, kinetic, regulatory and immunological properties of CPT of the midgut gland mitochondria of Macrobrachium borellii were assayed. CPT I optimum conditions were 34 °C and pH = 8.0. Kinetic analysis revealed a Km for carnitine of 2180 ± 281 μM and a Km for palmitoyl-CoA of 98.9 ± 8.9 μM, while V_max were 56.5 ± 6.6 and 36.7 ± 4.8 nmol min^− 1 mg protein^− 1, respectively. A Hill coefficient, n ~ 1, indicate a Michaelis–Menten behavior. The CPT I activity was sensitive to regulation by malonyl-CoA, with an IC₅₀ of 25.2 μM. Electrophoretic and immunological analyses showed that a 66 kDa protein with an isoelectric point of 5.1 cross-reacted with both rat liver and muscle-liver anti CPT I polyclonal antibodies, suggesting antigenic similarity with the rat enzymes. Although CPT I displayed kinetic differences with insect and vertebrates, prawn showed a high capacity for energy generation through β-oxidation of long-chain fatty acids.     

The routine metabolic rate of mulloway (Argyrosomus japonicus: Sciaenidae) and yellowtail kingfish (Seriola lalandi: Carangidae) acclimated to six different temperatures

This study compared the mass-specific routine metabolic rate (RMR) of similar sized mulloway (Argyrosomus japonicus), a sedentary species, and yellowtail kingfish (Seriola lalandi), a highly active species, acclimated at one of several temperatures ranging from 10–35 °C. Respirometry was carried out in an open-top static system and RMR corrected for seawater–atmosphere O₂ exchange using mass-balance equations. For both species RMR increased linearly with increasing temperature (T). RMR for mulloway was 5.78T − 29.0 mg O₂ kg^− 0.8 h^− 1 and for yellowtail kingfish was 12.11T − 39.40 mg O₂ kg^− 0.8 h^− 1. The factorial difference in RMR between mulloway and yellowtail kingfish ranged from 2.8 to 2.2 depending on temperature. The energetic cost of routine activity can be described as a function of temperature for mulloway as 1.93T − 9.68 kJ kg^− 0.8 day^− 1 and for yellowtail kingfish as 4.04T − 13.14 kJ kg^− 0.8 day^− 1. Over the full range of temperatures tested Q₁₀ values were approximately 2 for both species while Q₁₀ responses at each temperature increment varied considerably with mulloway and yellowtail kingfish displaying thermosensitivities indicative of each species respective niche habitat. RMR for mulloway was least thermally dependent at 28.5 °C and for yellowtail kingfish at 22.8 °C. Activation energies (E_a) calculated from Arrhenius plots were not significantly different between mulloway (47.6 kJ mol^− 1) and yellowtail kingfish (44.1 kJ mol^− 1).     

Effects of nitrite exposure on blood respiratory properties,acid-base and electrolyte regulation in the carp (Cyprinus carpio)

Summary Adult carp were subjected to 1 mM environmental nitrite for 48 h and nitrite uptake and changes in blood respiratory properties, extracellular electrolyte composition and acid-base status were examined.A constant influx of nitrite caused an accumulation of NO ₂ ^– in plasma to 5.4 mM in 48 h. The fraction of methaemoglobin rose with plasma [NO ₂ ^– ] to 83%, and the arterial oxygen content decreased to extremely low values. Arterial increased as a compensation to this O₂-shortage, whereas the O₂ saturation of the functional (unoxidized) haemoglobin decreased, revealing a reduction in its O₂ affinity.Blood haematocrit decreased as a result of red cell shrinkage, which caused very high red cell haemoglobin (Hb) concentrations. The erythrocytic nucleoside triphosphate (NTP) concentration showed a parallel increase whereby NTP/Hb, as well as the relative contributions of ATP and GTP to NTP, remained unchanged.Plasma [Cl^–] declined by 15 mM in 48 h, off-setting the plasma [NO ₂ ^– ] increase, minor changes in plasma [HCO ₃ ^– ] and a considerable increase in plasma [lactate]. Arterial pH and [HCO ₃ ^– ] rose slightly during the first 24 h of nitrite exposure, but returned to control values at 48 h. The rise in plasma [lactate] was not reflected in an extracellular metabolic acidosis. Plasma [K⁺] increased by 94% in 48 h, revealing an uncompensated extracellular hyperkalemia, whereas plasma [Na⁺] decreased, and plasma [Ca⁺⁺] was unchanged. Plasma osmolality remained essentially constant.The NO ₂ ^– accumulation could be reversed by transfer of the fish to NO ₂ ^– -free water, but nitrite off-loading was slower than the preceding NO ₂ ^– loading.Abbreviations Hb hemoglobin - NTP nucleoside triphosphate - Hct hematocrit - fractional saturation of Hb with oxygen     

Oxygen transfer properties and dimensions of red blood cells in high-altitude camelids,dromedary camel and goat

Summary To estimate the advantage of the small red blood cells (RBC) of high-altitude camelids for O₂ transfer, the kinetics of O₂ uptake into and release from the RBC obtained from llama, vicuña and alpaca were investigated at 37°C with a stopped-flow technique. O₂ transfer conductance of RBC (G) was estimated from the rate of O₂ saturation change and the corresponding O₂ pressure difference between medium and hemoglobin. For comparison, O₂ kinetics for the RBC of a lowaltitude camelid (dromedary camel) and the pygmy goat were determined and previously measured values for human RBC were used. O₂ transfer of RBC was found to be strongly influenced by extracellular diffusion, except with O₂ release into dithionite solutions of sufficiently high concentration (>30 mM). TheG values measured in these standard conditions,G _st (in mmol · min^–1 · Torr^–1 · (ml RBC)^–1) were: high-altitude camelids, 0.58 (averaged for llama, alpaca and vicuña since there were no significant interspecific differences); camel 0.42; goat, 0.42; man, 0.39. The differences can in part be attributed to expected effects of the size and shape of the RBC (volume, surface area, mean thickness), as well as to the intracellular O₂ diffusivity which depends on the concentration of cellular hemoglobin. The highG _st of RBC of highaltitude camelids may be considered to enhance O₂ transfer in lungs and tissues. But the O₂ transfer conductance of blood, , equal toG _st multiplied by hematocrit (in mmol · min^–1 · Torr^–1 · (ml blood)^–1), was only slightly higher as compared to other species: 0.20 (llama, alpaca, vicuña), 0.14 (camel), 0.18 (goat), 0.17 (man).Abbreviations DPG 2,3-diphosphoglycerate - G conductance - Hb hemoglobin - RBC red blood cells - percent saturation of hemoglobin     

The effect of feed restriction on expression of hepatic lipogenic genes in broiler chickens and the function of SREBP1

To study the role of sterol regulatory element-binding proteins (SREBP) in lipogenesis and cholesterol synthesis in the chicken, two experiments were carried out. In the first study, seven-week-old broilers (n = 16) were allocated into 2 groups, fasted for 24 h or refed for 5 h after a 24 h fasting. The mRNA concentrations for SREBPs and other lipogenic genes in the liver were determined by quantitative real time PCR. The hepatic mRNA relative abundance of lipogenic genes and genes involved in cholesterol synthesis were significantly greater (p < 0.001) in the refed broilers. Similar results were demonstrated with Northern analysis. The data suggest that in the liver of fasted broilers, genes associated with lipogenesis and cholesterol biosynthesis were inhibited. Indeed, the mRNA concentrations for fatty acid synthase (FAS), malic enzyme, and stearoyl coenzyme A desaturase were almost undetectable after the 24 h fasting. The data also demonstrated that the expression of lipogenic genes coordinate well as a group during the refeeding period. Second, three small interfering RNA (siRNA) oligonucleotides against SREBP1 were designed to be used in transfecting a chicken hepatocarcinoma cell line LMH. One of the three siRNAs effectively reduced SREBP1 mRNA concentration (p < 0.01). The acetyl coenzyme A carboxylase_α (ACC_α) mRNA was also significantly reduced by the SREBP1 siRNA treatment, suggesting that SREBP1 can upregulate the expression of this lipogenic gene. This siRNA, however, did not affect the mRNA for FAS. Taken together, the RNA interference study showed that SREBP1 has the ability to regulate the expression of ACC_α. This study has helped us understand more about the function of SREBP1 and the physiology of the broiler chickens.     

N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide

A complex trisaccharide β-d-GalpNAcA-(1 → 4)-β-d-GlcpNAc-(1 → 4)-d-ManpNAc (3) was prepared in a good yield (35%) in a transglycosylation reaction catalyzed by β-N-acetylhexosaminidase from Talaromyces flavus using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galacto-hexodialdo-1,5-pyranoside (1) as a donor followed by the in situ oxidation of the aldehyde functionality by NaClO₂. The disaccharide β-d-GlcpNAc-(1 → 4)-d-ManpNAc (2) was used as galactosyl acceptor. A disaccharide β-d-GalpNAcA-(1 → 4)-d-GlcpNAc (4; 39%) originated as a by-product in the reaction. Oligosaccharides comprising a carboxy moiety at C-6 are shown to be very efficient ligands to natural killer cell activation receptors, particularly to human receptor CD69. Thus, oxidized trisaccharide 3 is the best-known oligosaccharidic ligand to this receptor, with IC₅₀ = 2.5 × 10⁻⁹ M. The presented method of introducing a β-d-GalpNAcA moiety into carbohydrate structures is versatile and can be applied in the synthesis of other complex oligosaccharides.     

Synthesis, structural analysis and anticonvulsant activity of a ternary Cu(II) mononuclear complex containing 1,10-phenanthroline and the leading antiepileptic drug valproic acid

The synthesis and characterization of the binary complex of copper(II) with the antiepileptic drug valproic acid sodium salt (Valp) and the related ternary complex with 1,10-phenanthroline (phen) are reported, as well as the anticonvulsant properties of the latter. The characterization was carried out by means of elemental analyses, infrared (IR), UV–visible (UV–vis) spectrophotometry and Electron Paramagnetic Resonance (EPR). The X-ray crystal structure of the mononuclear complex bis(2-propylpentanoate)(1,10-phenanthroline)copper(II) [Cu(Valp)₂phen] is showed for the first time. It crystallized in C2/c space group with unit cell dimensions of a = 14.939(1) Å, b = 19.280(1) Å, c = 9.726(1) Å, β = 97.27(2)°, V = 2778.8(4) Å ³ and Z = 8. The carboxylates bond in an asymmetric chelating mode and the copper atom adopts a highly distorted octahedral coordination, characterized by the sum of the angles of 365.9° around Cu(II) and its nearest atoms in the CuN₂O₂ + O₂ chromophore instead of the expected 360° for a basal square planar geometry found in most Cu(II) complexes. Molecules assemble three by three through slipped π–π stacking of the aromatic phen with respectively 3.519 and 3.527 Å distances, in a head-to-tail arrangement. Studies of the anticonvulsant properties of this bioligand chelate evidenced its lack of efficacy in preventing MES-induced seizures. Interestingly, complex 4 protected mice against the Minimal Clonic seizures at doses that do not cause Rotorod toxicity, with an ED₅₀ documenting very potent anticonvulsant activity in this model of seizure, a particularly useful pharmacological profile of activity for the treatment of Petit Mal seizures.     

Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against β-glycosidases

β-(1→4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(β-d-Xylp)-4-thiohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(β-d-Galp)-4-thiopentopyranosid-2-ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced with NaBH₄ or K-Selectride, and the stereochemical course of the reduction was highly dependent on the reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH₄–THF at 0 °C gave a 3-deoxy-4-S-(β-d-Xylp)-4-thio-α-d-ribo-hexopyranoside derivative (6) as major product (74% yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11 gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-β-d-threo-pentopyranoside derivative 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at −78 °C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides 19 and 18. They displayed strong inhibitory activity against the β-galactosidase from Escherichia coli. Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (K_i = 0.80 mM), whereas 19 was a mixed-type inhibitor (K_i = 32 μM).     

Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: Role of p38 MAPK

Oxidative stress is characterized as an imbalance between the cellular production of oxidants and the cellular antioxidant defenses and contributes to the development of numerous cardiovascular and metabolic disorders, including hypertension and insulin resistance. The effects of prolonged oxidant stress in vitro on the insulin-dependent glucose transport system in mammalian skeletal muscle are not well understood. This study examined the in vitro effects of low-level oxidant stress (60–90 μM, H₂O₂) for 4 h on insulin-stimulated (5 mU/ml) glucose transport activity (2-deoxyglucose uptake) and on protein expression of critical insulin signaling factors (insulin receptor (IR), IR substrates IRS-1 and IRS-2, phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3)) in isolated soleus muscle of lean Zucker rats. This oxidant stress exposure caused significant (50%, p < 0.05) decreases in insulin-stimulated glucose transport activity that were associated with selective loss of IRS-1 (59%) and IRS-2 (33%) proteins, increased (64%) relative IRS-1 Ser³⁰⁷ phosphorylation, and decreased phosphorylation of Akt Ser⁴⁷³ (50%) and GSK-3β Ser⁹ (43%). Moreover, enhanced (37%) phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was observed. Selective inhibition of p38 MAPK (10 μM A304000) prevented a significant portion (29%) of the oxidant stress-induced loss of IRS-1 (but not IRS-2) protein and allowed partial recovery of the impaired insulin-stimulated glucose transport activity. These results indicate that in vitro oxidative stress in mammalian skeletal muscle leads to substantial insulin resistance of distal insulin signaling and glucose transport activity, associated with a selective loss of IRS-1 protein, in part due to a p38 MAPK-dependent mechanism.     

Subunit δ Is the Key Player for Assembly of the H+-translocating Unit of Escherichia coli FOF1 ATP Synthase

The ATP synthase (F_OF₁) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c₁₀γϵ and the stator ab₂α₃β₃δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F_OF₁ complexes by affinity chromatography with the use of mutants synthesizing different sets of F_OF₁ subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F_OF₁ is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H⁺-translocating unit as intermediate product. Surprisingly, during the biogenesis of F_OF₁, F₁ subunit δ is the key player in generating stable F_O. Subunit δ serves as clamp between ab₂ and c₁₀α₃β₃γϵ and guarantees that the open H⁺ channel is concomitantly assembled within coupled F_OF₁ to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H⁺-translocating enzymes.     

α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O₂ bond, explaining previous reports that AHSP decreases αHb O₂ affinity roughly 4-fold and promotes autooxidation due primarily to a 3–4-fold increase in the rate of O₂ dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O₂ dissociation rate and equilibrium constants, and reduced O₂-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when βHb is abundant but diverts αHb to a redox resistant holding state when βHb is limiting.     

Influence of chelators and iron ions on the production and degradation of H2O2 by β-amyloid–copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.