Glutamic Acid Decarboxylase in Sea Lamprey (Petromyzon marinus): Characterization, Localization, and Developmental Changes

Abstract: We have carried out assays for glutamic acid decarboxylase (GAD) in homogenates of brain and spinal cord from larval and adult sea lamprey ( Petromyzon marinus ). The enzyme had similar characteristics in both stages. Optimal pH was 6.8; optimal temperature was 27–30° C; K _m at 27°C was 5 mM. GAD activity was distributed uniformly along the length of the spinal cord. Specific activities for the larval cord and brain were 26 and 63 nm CO₂/mg protein/h. respectively. The specific activities for the adult cord and brain were 29 and 236 nm CO₂/mg protein/h, respectively. Thus, the activity of cord homogenates did not change significantly between larval and adult stages, but that of the brain increased about fourfold.     

Detection of ferryl myoglobin in the isolated ischemic rat heart

Reflectance spectroscopy was utilized to monitor the oxidation states of myoglobin (Mb) in isolated, buffer-perfused rat hearts. Hearts were subjected to 30 min global, no-flow ischemia, followed by reperfusion under anoxic conditions. The addition of Na₂S to the buffer at reperfusion permitted the detection of ferryl myoglobin (MbIV) as its sulfmyoglobin derivative. The accumulation of MbIV was prevented by addition of ascorbic acid (1 mM), ergothioneine (2mM), or desferal (1mM) to the buffer prior to ischemia. Ascorbate and other agents have been previously shown to serve as one-electron reductants of MbIV. We propose that during the early phases of ischemia, deoxymyoglobin is oxidized to MbIV by residual H₂O₂. It also seems reasonable that the peroxidative activity of Mb(IV), during oxygenated reperfusion, might lead to cellular damage if this hypervalent form of Mb is not reduced.     

Recombinant expression of Mus musculus myoglobin

The cDNA encoding for Mus musculus myoglobin (Mb) was amplified using standard RT-PCR techniques and cloned in an appropriate bacterial expression vector. For the first time, mouse Mb was recombinantly expressed in Escherichia coli cells, BL21(DE3), and purified in sufficient amounts to carry out a preliminary characterization. As shown by mass spectrometry, the protein is found in complex with glutathione, which binds the Cys residue in the topological position E9, in the proximity of the heme group. In recombinant murine Mb, azide affinities are only slightly dependent on the Cys(E9) oxidation state. This suggests that Cys(E9) does not provide a relevant contribution for the stabilization of ligands bound to the heme iron atom. Recombinant expression of M. musculus Mb might have an important role in order to investigate the eventual involvement of Cys(E9) in the new physiological roles proposed for Mb.     

Acetylcholinesterase from the Skeletal Muscle of the Lamprey Petromyzon marinus Exists in Globular and Asymmetric Forms

To obtain information about the evolution of acetylcholinesterase (AChE), we undertook a study of the enzyme from the skeletal muscle of the lamprey Petromyzon marinus, a primitive vertebrate. We found that the cholinesterase activity of lamprey muscle is due to AChE, not pseudocholinesterase; the enzyme was inhibited by 1,5-bis(4-allyldimethylammonium phenyl) pentane-3-one (BW284C51), but not by tetramonoisopropyl pyrophosphortetramide (iso-OMPA) or ethopropazine. Also, the enzyme had a high affinity for acetylthiocholine and was inhibited by high concentrations of substrate. A large fraction of the AChE was found to be glycoprotein, since it was precipitated by concanavalin A-agarose. Optimal extraction of AChE was obtained in a high-salt detergent-containing buffer; fractional amounts of enzyme were extracted in buffers lacking salt and/or detergent. These data suggest that globular and asymmetric forms of AChE are present. On sucrose gradients, enzyme that was extracted in high-salt detergent-containing buffer sedimented as a broad peak of activity corresponding to G4; additionally, there was usually a peak corresponding to A12. Sequential extraction of AChE in conjunction with velocity sedimentation resolved minor forms of AChE and revealed that the G1, G2, G4, A4, A8, and A12 forms of AChE could be obtained from the muscle. The identity of the forms was confirmed through high-salt precipitation and collagenase digestion. The asymmetric forms of AChE were precipitated in low ionic strength buffer, and their sedimentation coefficients were shifted to higher values by collagenase digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of immunoreactive myosin and myoglobin in the developing chicken gizzard

Summary Antibodies to adult-type myosin and myoglobin from chicken gizzard were used to study the expression of these proteins during chicken embryogenesis. Using the indirect immunofluorescent technique, myosin was detected as discrete fluorescent foci in the central part of the presumptive chicken gizzard as early as day 5 of development. During the following days, immunoreactive myosin extends both craniocaudally as well as laterally and reaches the serosal and luminal borders by day 13/14. On day 16, the adult fascicular pattern is achieved. As judged by enzymelinked immunoassay and spectroscopic methods, myoglobin did not appear until day 18.Dedicated to Mrs. C.F. Schoenberg, Department of Anatomy, Cambridge, Great Britain     

Testosterone stimulates myoglobin expression in different muscles of the mouse

The regulation of energy metabolism is one of the major functions of steroid hormones. This study was performed to explore whether testosterone can regulate the aerobic capacity of skeletal muscles via myoglobin expression. To study this, changes in testosterone level were quantified, and the level of myoglobin protein was analyzed using Western blot in mice subjected to 6 weeks of training (T) or testosterone administration (A). Both treatments significantly increased the plasma testosterone level when compared to the untrained (U) or control (C) group. Training induced a significant increase in the myoglobin content in gastrocnemius and plantaris muscles (287 and 83%, respectively). Testosterone administration increased myoglobin concentration in plantaris (183%) but not in gastrocnemius. In extensor digitorum longus muscle the protein content decreased slightly after exercise, but increased 78% after testosterone administration. In soleus and rectus femoris muscles the myoglobin content was unchanged after both treatments. The data show that testosterone and training have differential effects on the concentration of myoglobin in some, but not all muscles. This may have an influence on the aerobic capacity in mouse skeletal muscles. The data demonstrated that both testosterone administration and training induced an increase in plasma testosterone level. However, the effects of the treatments on the myoglobin concentration differ.     

Lampetra (Eudontomyzon) gracilis,a synonym of Eudontomyzon danfordi

Synopsis Evidence is presented which suggests that the nonparasitic lamprey, Lampetra (Eudontomyzon) gracilis Kux, 1965, is conspecific with the parasitic lamprey Eudontomyzon danfordi Regan, 1911. The diagnostic characters of the holotype and of the non-type material of E. gracilis are features found in E. danfordi specimens in their second and final year of adult life, thereby making the former a junior synonym of the latter.     

Oxygen consumption by ammocoetes of the lampreyGeotria australis in air

 When covered by moistened lint-free gauze, the larvae (ammocoetes) of the lamprey Geotria australis survived, without apparent discomfort, for 4 days in water-saturated air at 10, 15 and 20 °C. In air, the mean standard rates of O₂ consumption of medium to large ammocoetes of G. australis (xˉ=0.52 g) at 10, 15 and 20 °C were 14.5, 35.7 and 52.1 μl⋅g^-1⋅h^-1, respectively. At 15 °C, the slope of the relationship between log O₂ consumption (μl O₂⋅h^-1) and log body weight for ammocoetes over a wide range in body weight was 0.987. The Q ₁₀s for rate of O₂ consumption between 10 and 15 °C, 15 and 20 °C and 10 and 20 °C were 4.9, 2.9 and 3.6, respectively. Our results and observations of the ammocoetes suggest that, when out of water, larval G. australis derives most of its O₂ requirements from cutaneous respiration, particularly at lower temperatures. This would be facilitated by the small size and elongate shape (and thus a relatively high surface-to-volume ratio), low metabolic rate, thin dermis, extensive subdermal capillary network and high haemoglobin concentration of larval G. australis. Accepted: 28 March 1996     

Immunocytochemical study of fibronectin in the sea lamprey,Petromyzon marinus,and the Atlantic hagfish,Myxine glutinosa

Summary Immunoreactive fibronectin-like material was localized within tissues of agnathans (hagfishes and lampreys) by an immunoperoxidase technique. Fibronectin was detected in basement membranes and in loose and dense connective tissues throughout the agnathan body. A fibronectin-like component was also identified in the plasma of both lampreys and hagfishes. The results indicate that fibronectin or a fibronectin-like material is a major component of agnathan connective tissues. Although there were some variations in the localization of fibronectin both between the lamprey and the hagfish and between agnathan and other vertebrate tissues, the generalized pattern of distribution of fibronectin in the agnathans supports the view that this protein, like that in higher vertebrates, plays a role in cellmatrix adhesion and tissue organization.     

XANES study of iron displacement in the haem of myoglobin

The XANES (X-ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES between HbA and Mb are assigned to changes in the Fe-porphyrin geometry.     

Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

The role of entropy in the discrimination between CO and O2 in myoglobin

Using stopped-flow rapid mixing and flash photolysis techniques, the dissociation rate coefficients of horse carbonmonoxy myoglobin (hMbCO) and oxygenated myoglobin (hMbO₂) in aqueous solution have been determined as a function of temperature between 274 and 342 K. From the Arrhenius plot, an activation enthalpy for dissociation of 74 kJ/mol was obtained for both ligands. The pronounced kinetic differences arise from markedly different pre-exponentials. We compare the Arrhenius parameters with those of the association reaction, as measured at cryogenic temperatures. In our analysis we conclude that the entropy loss upon binding of O₂ is twice as large as that for CO. Taking reasonable estimates for the frequency factor, the transition state entropy in hMbO₂ is located roughly half way in between the entropies of the bound and unbound states. By contrast, the entropy of the transition state in hMbCO appears to be identical to that of the bound state. Possible structural reasons for the different behavior are discussed. Received: 13 January 1997 / Accepted: 24 April 1997     

An exceptional amino acid replacement on the distal side of the iron atom in proboscidean myoglobin

Summary Amino acid sequence determination of elephant myoglobin revealed the presence of the unusual substitution E7 His Gln. Stereochemical analyses suggest that the most suitable residue which can functionally substitute for His at this position in vertebrate globins is Gln. Physiochemical studies imply that the slower rate of autooxidation of elephant myoglobin is the result of this substitution which may confer some selective advantage on the species. Comparative sequence data of paenungulate myoglobins suggest that the His Gln mutation probably occurred in an ancestor of Elephantinae.     

Solvent modulation of the structural heterogeneity in FeIII myoglobin samples: a low temperature EPR investigation

High spin FeIII myoglobin samples in solutions with different solvent composition have been investigated at low temperature by Electron Paramagnetic Resonance spectroscopy. The g = 6 line of the spectrum has been analyzed in terms of a distribution of the two crystal field parameters ₁ and ₂. By means of the Angular Overlap Method, it has been shown that these distributions entail, in turn, a distribution in the iron-heme displacement along the normal to the heme-plane. The spread in this iron-heme distance, which can be connected with the binding action of the proximal histidine, has been proposed as a quantitative measurement of the structural heterogeneity (conformational substate landscape) displayed by the protein molecules. The results point out, moreover, that the solvent composition can affect the structural heterogeneity of the protein system. In particular, addition of glycerol, ethylene glycol and sucrose yields a significant reduction in the spread of the ironheme displacement, while the presence of ammonium sulfate induces a change in the average position of the iron in the heme-plane. The role played by the solvent in the structure and dynamics of the protein, in connection also with the conformational substate distribution, is discussed. Correspondence to: S. Cannistraro     

In vitro nonenzymatic glycation enhances the role of myoglobin as a source of oxidative stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO₂ and GMbO₂, respectively. H₂O₂-induced iron release was significantly higher from GMbO₂ than that from MbO₂. This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H₂O₂ was studied. GMbO₂ degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO₂. Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H₂O₂ with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO₂. Compared to MbO₂, GMbO₂ was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO₂. GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H₂O₂. These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

The structural dynamics of myoglobin

Conformational fluctuations in proteins were initially invoked to explain the observation that diffusion of small ligands through the matrix is a global phenomenon. Small globular proteins contain internal cavities that play a role not only in matrix dynamics but also in controlling function, tracing a pathway for the diffusion of the ligand to and from the active site. This is the main point addressed in this Review, which presents pertinent information obtained on myoglobin (Mb). Mb, a simple globular heme protein which binds reversibly oxygen and other ligands. The bond between the heme Fe(II) and gaseous ligands can be photodissociated by a laser pulse, generating a non-equilibrium population of protein structures that relaxes on a picosecond to millisecond time range. This process is associated with migration of the ligand to internal cavities of the protein, which are known to bind xenon. Some of the results obtained by laser photolysis, molecular dynamics simulations, and X-ray diffraction of intermediate states of wild-type and mutant myoglobins are summarized. The extended relaxation of the globin moiety directly observed by Laue crystallography reflects re-equilibration among conformational substates known to play an essential role in controlling protein function.     

Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin

Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the alpha-gamma axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the alpha-gamma axis without leaving the "protein cage."     

Bacterial expression and characterization of molluscan IDO-like myoglobin

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic α- and β-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His⁷⁴, His²⁸⁸, and His³³² were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His³³² is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His⁷⁴ and His²⁸⁸ might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.