Nature of the FeO2 bonding in myoglobin: an overview from physical to clinical biochemistry

The iron(II)-dioxygen bond in myoglobin and hemoglobin is a subject of wide interest. Studies range from examinations of physical-chemical properties dependent on electronic structure, to investigations of stability as a function of oxygen supply. Stability properties are of particular importance in vivo, since the oxygenated form is known to be oxidized easily to the ferric form, which cannot be oxygenated and is therefore physiologically inactive. Kinetic and thermodynamic studies of the stability of native oxymyoglobin have revealed a new feature in FeO2 bonding. In vivo, the iron center is always subject to a nucleophilic attack of the water molecule or hydroxyl ion, which can enter the heme pocket from the surrounding solvent, and thereby irreversibly displace the bound dioxygen from MbO2 in the form of O2- so that the iron is converted to the ferric form. A free energy diagram for the potential reactions of FeO2 visualizes myoglobin as a molecular structure that can provide in solution the delicate balance of kinetic and thermodynamic factors necessary to stabilize reversible oxygenation, as opposed to irreversible autoxidation to metmyoglobin.     

The dimeric and co-operative myoglobin ofNassa mutabilis. A peculiar case

The myoglobin present in the radular muscle of the Prosobranchia sea snailNassa mutabilis is a peculiar case among myoglobins. It is a dimer showing co-operative oxygen binding equilibrium curves with pO_{2 1/2}=4.7 mmHg, invariant with pH, and n=1.6. Although the globin is composed of 147 amino acid residues, corresponding to a molecular mass of 15760 D, gel filtration chromatography of the native myoglobin indicate Mr=26000±2000 D. Similarly, acrylamide electrophoretic analyses in SDS and velocity sedimentation indicate a molecular mass of about 13000 D for the denatured globin. The molecule is highly unstable and forms slowly a chromogen when aged or immediately upon oxidation to the ferric state. The visible region of the absorption spectrum of the O₂ or CO liganded myoglobin derivatives indicate an altered heme environment. Circular dichroism analyses confirm this indication showing negative Cotton effects in all regions of the heme absorption spectra of the MbO₂ and MbCO derivatives. Interestingly, the CD spectrum of the oxidised met-form shows a positive band almost symmetrical with respect to that of the MbO₂ derivative. This is similar to what reported for the monomeric hemoglobin ofGlycera dibranchiata for which a reversed heme orientation was proposed. Detailed resonance Raman spectroscopic studies have permitted a more direct investigation of the interactions between the heme and the protein. The proximal Fe-Im bond shows a stretching mode frequency down shifted by 5 cm^–1 with respect to the corresponding band of horse heart myoglobin, in good correlation with the much higher instability ofNassa m. myoglobin and its much lower oxygen affinity. The unusual bond instability finds additional support in a kinetic study in which the myoblogin is mixed with CO in buffered solutions at different pH values. This approach gives evidence that the Fe-Im bond is broken upon lowering the pH, with a pK of 4.0±0.2, the highest among those of deoxy hemoproteins. The rupture of the proximal bond appears to occur with a proton-linked transition showing n=1.8±0.1, again indicating cooperativity between the two subunits. The vinyl and propionate heme substituents show resonance Raman spectroscopic bands indicating different modes of interaction with their environment with respect to other myoglobins. Most interestingly, the vinyl stretching mode frequency, typically a single band, appears split in two bands inNassa m. myoglobin. This splitting is evident in all the investigated derivatives of the myoglobin, indicating that vinyl 2 and 4 are not equivalent in this molecule. A similar splitting has been found so far only inChironomus t.t. hemoglobin.     

Effect of 15(R)-15-methyl-prostaglandin E2 on iron absorption in the rat

Summary The administration of 15(R)-15-methyl prostaglandin E₂ (15(R)-15-M-PGE₂) in vivo significantly diminished the uptake of⁵⁹Fe into blood, spleen, liver, femur and dried intestine of rats, whereas acetylsalicylic acid (ASA) increased the counts significantly. This effect of ASA was counteracted by 15(R)-15-M-PGE₂. It is suggested that prostaglandins (PGs) might play an important role in inhibiting iron absorption at the intestinal level.This work was supported by grant No.6638 from CONICET (Argentina). The technical assistance of Mrs María E. Castro and Norma Rizzo is gratefully acknowleged.     

The action of the peptidoleukotriene LTD4 on intracellular calcium in rat mesangial cells

The dose-dependent effect of CGP 45715A on the LTD₄-induced Ca²⁺ response of glomerular mesangial cells has been studied. Our results demonstrate that the LTD₄-dependent increase in the cytosolic Ca²⁺ concentration primarily involves an InsP₃-mediated release of Ca²⁺ from intracellular storage sites and to a minor extent an enhanced influx of Ca²⁺ through receptor-operated Ca²⁺ channels located in the plasma membrane. The action of CGP 45715A on the Ca²⁺ response is an inhibitory one and is convincingly explained by a displacement of LTD₄ from its receptor site(s). The contractile effect of LTD₄ on pulmonary smooth muscle is proposed to be mainly caused by a receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate.     

Differential vascular effects of neuropeptide Y(NPY) selective receptor agonists

Neuropeptide Y (NPY) increases blood pressure either directly or indirectly by potentiating the effect of various vasoconstrictors. Only one (the Y₁-receptor) of two subtypes of receptors (Y₁ and Y₂) is thought to mediate the vascular smooth muscle contraction. To test this hypothesis we challenged isolated rat mesenteric arteries that had a functional endothelium with (1–36) NPY and with specific Y₁-receptor ([Leu³¹, Pro³⁴] NPY) and Y₂-receptor ([Ahx^5–24, -Glu²--Lys³⁰] NPY) agonists. The Y₁-receptor agonist elicited a contractile response similar to that of NPY, whereas the Y₂-receptor agonist had no effect on wall tension. We also found that the presence of a functional endothelium has no influence on the contractile response to NPY. From these data we conclude that the direct contractile effect of NPY in the mesenteric artery is mediated by stimulation of Y₁-receptors and is not endothelium-dependent.     

Influence of environmental conditions on the amount of N2O released from activated sludge in a domestic waste water treatment plant

Waste water purification is characterized by intensive mineralization and nitrification processes. Because of the high O₂ demand, temporarily anaerobic conditions may be produced, and denitrification by nitrifying organisms as well as heterotropic denitrification may contribute to N₂O release. In situ measurements (1993–1994) suggest that N₂O is released from activated sludge in a domestic waste water treatment plant at an average rate of 1040 g m^–2h^–1 with a range between zero and 6198 g m^–2h^–1. The production of N₂O seems to be related to the concentration of NO ₂ ^– and NO ₃ ^– as well as to the pH. In the waste water about 75–200 g N₂O l^–1 is dissolved. This N₂O is released after discharge into the receiving waters. The N₂O is produced essentially by nitrification rather than by heterotropic denitrification. On a long-term scale the increasing use of mechanical-biological waste water purification plants world-wide may add increasingly to the anthropogenic production of N₂O, although the present amount of N₂O produced is negligible compared to its global terrestrial production.     

Respiratory burst in peritoneal exudate cells in response to a modified tuftsin

Intraperitoneal administration of tuftsin-M [Thr–Lys–Pro–Arg–NH–(CH₂)₂–NH–CO–C₁₅H₃₁] to Balb/C mice has been shown to induce a respiratory burst in the peritoneal exudate cells. The macrophages exhibited enhanced levels of O₂ ^–, H₂O₂, NADPH oxidase and myeloperoxidase, but the activities of superoxide dismutase, catalase and glutathione peroxidase remained virtually unchanged. The magnitude of the oxidative burst depended directly on the dose of tuftsin-M; higher activity was observed at higher doses of the peptide. Tuftsin-M enhanced the generation of both O ₂ ^– and H₂O₂ under in vitro conditions, as did phorbol myristate acetate. These results suggest that tuftsin-M could enhance non-specific defence against infections by activating the macrophages.     

Role of adenosine A1 and A2 receptors in the regulation of aldosterone production in rat adrenal glands

Summary To investigate the roles of adenosine A₁ and A₂ receptors in the regulation of aldosterone production, we examined the effects of adenosine and adenosine agonists (N⁶-cyclohexyl adenosine; selective adenosine A₁ receptor agonist and 5-N-ethylcarboxamine adenosine; selective adenosine A₂ receptor agonist) on aldosterone and cyclic AMP production in rat adrenal capsular cells. Neither adenosine nor 5-N-ethylcarboxamine adenosine caused significant effects on basal aldosterone or cyclic AMP production. Also, adenosine (10^–3M) showed no consistent effects on aldosterone and cyclic AMP production induced by ACTH. On the other hand, N⁶-cyclohexyl adenosine exhibited a significant inhibition of basal aldosterone and cyclic AMP production at doses of 10^–4 M and 10^–3 M; furthermore, 10^–3 M N⁶-cyclohexyl adenosine inhibited aldosterone and cyclic AMP production stimulated by ACTH. These results suggest that adenosine A₁ receptors are coupled to and inhibit adenylate cyclase and may be involved in the inhibition of aldosterone production.     

Invertebrate neuropeptides resembling vasotocin and some analogues: Synthesis and pharmacological properties

Summary The solid phase synthesis of three invertebrate vasopressin-oxytocin homologs: AVP-like factor, F₁ ¹, ([Leu², Thr⁴] AVT)² isolated from subesophageal and thoracic ganglia ofLocusta migratoria ³, Arg-conopressin-S⁴. ([Ile², Arg⁴] AVT), Lys-conopressin-G⁴ ([Phe², Arg⁴] LVT), both isolated from the venom of fish-hunting marine snails of the genusConus and six of their analogues is reported. These analogues are: [Arg⁴] AVT, [Ile²] AVT, [Leu²] AVT, [Phe², Arg⁴] AVT, [Arg⁴] LVT and [Ile², Arg⁴] LVT. All peptides were tested for antidiuretic and vasopressor activities.     

Arachidonic acid signaling is involved in the mechanism of imidazoline-induced K<Subscript>ATP</Subscript> channel-independent stimulation of insulin secretion

The mechanism by which the novel, pure glucose-dependent insulinotropic, imidazoline derivative BL11282 promotes insulin secretion in pancreatic islets has been investigated. The roles of K_ATP channels, α₂-adrenoreceptors, the I₁-receptor-phosphatidylcholine-specific phospholipase (PC-PLC) pathway and arachidonic acid signaling in BL11282 potentiation of insulin secretion in pancreatic islets were studied. Using SUR1^(-/-) deficient mice, the previous notion that the insulinotropic activity of BL11282 is not related to its interaction with K_ATP channels was confirmed. Insulinotropic activity of BL11282 was not related to its effect on α₂-adrenoreceptors, I₁-imidazoline receptors or PC-PLC. BL11282 significantly increased [³H]arachidonic acid production. This effect was abolished in the presence of the iPLA₂ inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, which is independent of concomitant changes in cytoplasmic free Ca²⁺ concentration, involves release of arachidonic acid by iPLA₂ and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway. Received 5 July 2007; received after revision 18 September 2007; accepted 20 September 2007     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Alkalosis and renal excretion of ammonia by rat kidney

Summary Upon sulfate administration, U_pH falls more in alkalotic rats than in controls. Alkalosis can lead to a reduction in U_{NH 3}V at highly acidic urine. The significance of this process is doubtful at U_pH ranging from about 6 to 7. At lower U_pH less NH₃ would be excreted, thereby less H⁺ would be trapped in urine and some acid would be conserved.     

Hypotensive activity of histidine-containing analogues of C-terminal hexapeptide of Substance P

Summary Four new hexapeptide analogues of C-terminal Substance P fragment with increased solubility in aqueous solutions are described. The peptides contain histidine in positions 6, 8, 9 and 10, respectively. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of amino acid residues in various positions in the C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive and antigenic properties, respectively. Only the [His⁶] SP_6-11 analogue had an unchanged antigenic structure when compared with the C-terminal region of Substance P, but it showed an almost total loss of hypotensive activity. The [His⁹] SP_6-11 analogue retained 50% of the hypotensive activity of the C-terminal hexapeptide but showed a markedly reduced expression of the antigenic epitope localized in this region of Substance P.     

Stability analysis of the bacteriophage ϕKMV lysin gp36C and its putative role during infection

The kinetic, thermodynamic and structural stability of gp36C, the virion-associated peptidoglycan hydrolase domain of bacteriophage ϕKMV, is analyzed. Recombinant gp36C is highly thermoresistant (k = 0.595 h⁻¹ at 95°C), but not thermostable (T_m = 50.2°C, ΔH_cal = 6.86 × 10⁴ cal mol⁻¹). However, aggregation influences kinetic stability in an unusual manner since aggregation is more pronounced at 55°C than at higher temperatures. Furthermore, gp36C reversibly unfolds in a two-state endothermic transition, and circular dichroism analysis shows that gp36C almost completely refolds after a 3-h heat treatment at 85°C. These properties are in agreement with gp36C being part of the extensible tail which is ejected in an unfolded state during phage infection. Received 24 April 2006; received after revision 26 May 2006; accepted 10 June 2006     

VIP and histamine H2 receptor activity in human fetal gastric glands

Summary Vasoactive intestinal peptide (VIP, EC₅₀=6.4×10^–10 M) and histamine (EC₅₀=3×10^–6 M) activated the cyclic AMP generating system in gastric glands isolated from two human fetuses at 23 weeks gestation. Histamine antagonism by the H₂ receptor blockers cimetidine (Ki=0.35×10^–6 M) and ranitidine (ki=0.51×10^–7 M) clearly characterized the histaminic activation as being of the H₂ type. It is suggested that these two vasoactive hormones may operate as neurocrine/paracrine regulators of the differentiation and/or function of the human gastric mucosa in utero.     

Evidence of a direct action of triiodothyronine (T3) on the cell membrane of GH3 cells: an electrophysiological approach

Summary Electrophysiological experiments demonstrate that triiodothyronine (T₃) exerts a direct effect on the membrane of a strain of cultured rat pituitary tumor cells, GH₃/B₆. These cells respond to pressure application of T₃ (2–5 nl, concentration 1·10^–10 M) with an increase in the membrane resistance (R_m) and a hyperpolarization. Spontaneously firing cells become silent.     

Malignant hyperthermia: Molecular defects in membrane permeability

Summary Malignant hyperthermia (MH), a genetically inherited disorder of skeletal muscle, is due to molecular defect in membrane permeability. The alteration in membrane permeability is suggested to be due to enhanced phospholipase A₂ activity which is responsible for the increased level in sarcoplasmic Ca²⁺. The excess Ca²⁺ is responsible for muscle hyper-rigidity and enhanced rate of glycolysis, resulting in a rapid rate of lactic acid production and a low pH in MH muscle.     

Diabetes alters drug metabolism-in vivo studies in a streptozotozin-diabetic rat model

Summary The influence of experimental streptozotozin-induced diabetes on hepatic drug metabolism in vivo has been studied in rats, using¹⁴CO₂-exhalation after¹⁴C-aminopyrine injection. Male diabetic rats showed a decreased (–18%), females an increased (+19%)¹⁴CO₂-exhalation compared to controls, indicating altered hepatic drug metabolism due to diabetes.     

Effect of xanthurenic acid on P-450-dependent biotransformation by molting glands in vitro

Incubation of molting glands from the crayfishProcambarus clarkii (Y-organ) and the silkwormBombyx mori (prothoracic gland) with 23,24-[²H₄]-2-deoxyecdysone resulted in the production of deutero-ecdysone; this biotransformation was inhibited in the presence of xanthurenic acid. When the experiments were performed under an¹⁸O₂ atmosphere, the¹⁸O atom was introduced into ecdysone, as confirmed by mass spectrometry. We therefore suggest that xanthurenic acid inhibits P-450-dependent hydroxylation of 2-deoxyecdysone. However, deutero-2-deoxyecdysone was not converted to 3-dehydroecdysone when using Y-organs in vitro, although it is a major product. We therefore conclude that the biosynthetic pathway of ecdysteroids inP. clarkii branches at an early step.     

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action

A large variety of snake toxins evolved from PLA₂ digestive enzymes through a process of ‘accelerated evolution’. These toxins have different tissue targets, membrane receptors and mechanisms of alteration of the cell plasma membrane. Two of the most commonly induced effects by venom PLA₂s are neurotoxicity and myotoxicity. Here, we will discuss how these snake toxins achieve a similar cellular lesion, which is evolutionarily highly conserved, despite the differences listed above. They cause an initial plasma membrane perturbation which promotes a large increase of the cytosolic Ca²⁺ concentration leading to cell degeneration, following modes that we discuss in detail for muscle cells and for the neuromuscular junction. The different systemic pathophysiological consequences caused by these toxins are not due to different mechanisms of cell toxicity, but to the intrinsic anatomical and physiological properties of the targeted tissues and cells. Received 05 March 2008; received after revision 08 April 2008; accepted 29 April 2008