RESUMO
The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.
Assuntos
Cartilagem/análise , Colágeno/análise , Acetatos , Ácido Acético , Animais , Cartilagem/ultraestrutura , Embrião de Galinha , Colágeno/imunologia , Epitopos/análise , Imuno-Histoquímica , Microscopia Eletrônica , Pepsina A , TripsinaRESUMO
Chondrocytes in dense suspension culture in agarose survive in serum-free DME because they secrete low molecular mass compounds supporting their own viability. This activity can be replaced by pyruvate, or sulfhydryl compounds, e.g., cysteine or dithioerythritol. Catalase, an enzyme decomposing H2O2, also protects the cells, whereas superoxide dismutase has no effect. Therefore, chondrocytes in culture are sensitive to toxic compounds derived from molecular oxygen, i.e., hydroxyl radicals or hydrogen peroxide spontaneously generated in DME containing ascorbate and ferrous ions. Poly-ADP-ribosylation is an important step in the cascade of events triggered by these compounds. To survive, chondrocytes do not require stimulation by growth factors. They remain resting cells in fully defined, serum-free culture also at low density. Proliferation and hypertrophy can be induced by serum, but not by low cell density alone.
Assuntos
Cartilagem/citologia , Animais , Benzamidas/farmacologia , Cartilagem/efeitos dos fármacos , Catalase/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Colágeno/biossíntese , Colágeno/isolamento & purificação , Meios de Cultura , Cisteína/farmacologia , Cinética , Piruvatos/farmacologia , Superóxido Dismutase/farmacologiaRESUMO
The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.
Assuntos
Cartilagem/ultraestrutura , Colágeno/fisiologia , Matriz Extracelular/ultraestrutura , Animais , Embrião de Galinha , Imunofluorescência , Ouro , Microscopia Eletrônica/métodos , Distribuição TecidualRESUMO
In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.
Assuntos
Cartilagem/citologia , Colágeno/biossíntese , Fator de Crescimento Insulin-Like I/farmacologia , Insulina/farmacologia , Tiroxina/farmacologia , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Meios de Cultura Livres de Soro , Sangue Fetal , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteoglicanas/biossínteseRESUMO
It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.
Assuntos
Cartilagem/análise , Colágeno/análise , Animais , Cartilagem/ultraestrutura , Centrifugação , Embrião de Galinha , Eletroforese em Gel de Poliacrilamida , Microscopia EletrônicaRESUMO
Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.
Assuntos
Cartilagem/citologia , Animais , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Divisão Celular , Células Cultivadas , Embrião de Galinha , Colágeno/biossíntese , Meios de Cultura , DNA/análise , Replicação do DNA , Cinética , Microscopia Eletrônica , Proteoglicanas/biossíntese , SefaroseRESUMO
Adhesive interactions between neurons and extracellular matrix (ECM) play a key role in neuronal pattern formation. The prominent role played by the extracellular matrix protein tenascin/cytotactin in the development of the nervous system, tied to its abundance, led us to speculate that brain may contain yet unidentified tenascin receptors. Here we show that the neuronal cell adhesion molecule contactin/F11, a member of the immunoglobulin(Ig)-superfamily, is a cell surface ligand for tenascin in the nervous system. Through affinity chromatography of membrane glycoproteins from chick brain on tenascin-Sepharose, we isolated a major cell surface ligand of 135 kD which we identified as contactin/F11 by NH2-terminal sequencing. The binding specificity between contactin/F11 and tenascin was demonstrated in solid-phase assays. Binding of immunopurified 125I-labeled contactin/F11 to immobilized tenascin is completely inhibited by the addition of soluble tenascin or contactin/F11, but not by fibronectin. When the fractionated isoforms of tenascin were used as substrates, contactin/F11 bound preferentially to the 190-kD isoform. This isoform differs in having no alternatively spliced fibronectin type III domains. Our results imply that the introduction of these additional domains in some way disrupts the contactin/F11 binding site on tenascin. To localize the binding site on contactin/F11, proteolytic fragments were generated and characterized by NH2-terminal sequencing. The smallest contactin/F11 fragment which binds tenascin is 45 kD and also begins with the contactin/F11 NH2-terminal sequence. This implies that contactin/F11 binds to tenascin through a site within the first three Ig-domains.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/química , Embrião de Galinha , Cromatografia de Afinidade , Contactinas , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/química , Imunoglobulinas/química , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Fragmentos de Peptídeos/metabolismo , Ensaio Radioligante , TenascinaRESUMO
A number of mutant hemoglobins are inordinately unstable, denaturing in circulating red cells into Heinz bodies, resulting in congenital Heinz body hemolytic anemia (CHBHA). We have emphasized that most such hemoglobins involve amino acid substitutions at sites neighboring the heme group of the beta-polypeptide chain, and have shown that heme binding to globin is diminished thereby. Thus, hemes were progressively lost from four unstable hemoglobins (Köln, Hammersmith, San Francisco, and Zürich) as they precipitated into Heinz bodies at 50 degrees C. The role of heme loss, especially from beta chains, in Heinz body formation was supported by studies with a hemoglobin synthesized to contain hemes only on its alpha chains (alpha(2) (heme)beta(2) (0)). The behavior of this compound, postulated to be an intermediary in the formation of Heinz bodies, mimicked that of the genetically unstable hemoglobins in several ways: (a) it precipitated at 50 degrees C into typical coccoid Heinz bodies; (b) as also observed with CHBHA hemoglobins this denaturation was virtually prevented by the heme ligands, cyanide or carbon monoxide, which inhibit further heme loss; it was potentiated by oxidation of hemes to the ferri- state, which accentuates heme loss; (c) the thiol groups of alpha(2) (heme)beta(2) (0) were hyperreactive, forming mixed disulfides with glutathione and membrane sulfhydryls at rates similar to those of CHBHA hemoglobins and 10 or more times that of normal hemoglobin A; (d) heme repletion of the protein molecules by the addition of crystalline hemin to either alpha(2) (heme)beta(2) (0) or to the genetically unstable hemoglobins, prevented their precipitation into Heinz bodies and normalized their aberrant electrophoretic behaviors; and (e) during Heinz body formation at 50 degrees C both alpha(2) (heme)beta(2) (0) and the genetically unstable hemoglobins released free alpha(heme)-chains into solution, suggesting that the bulk of the whitish, Heinz body precipitate is naked beta(8)-chains. We conclude that heme loss from mutant beta chains is an early step in Heinz body formation in several of the unstable hemoglobinopathies. The resulting hemedepleted compounds, of which synthetic alpha(2) (heme)beta(2) (0) is a prototype, are unstable, cleaving into beta(0)-chain precipitates (the bulk of the Heinz body material) and soluble, free alpha(heme)-chains (demonstrated previously in hemolysates from many patients with CHBHA).
Assuntos
Anemia Hemolítica Congênita/sangue , Corpos de Heinz , Heme , Hemoglobinas Anormais , Sítios de Ligação , Eletroforese das Proteínas Sanguíneas , Monóxido de Carbono/farmacologia , Cianetos/farmacologia , Glutationa/metabolismo , Hemoglobinas Anormais/metabolismo , Histidina/metabolismo , Temperatura Alta , Humanos , Métodos , Peptídeos/metabolismo , Ligação Proteica , Compostos de Sulfidrila/metabolismoRESUMO
BACKGROUND: . beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now. RESULTS: . The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively. CONCLUSIONS: . Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.
Assuntos
Actinomycetales/enzimologia , Manosidases/química , Sequência de Aminoácidos , Sequência de Carboidratos , Catálise , Estabilidade Enzimática , Temperatura Alta , Manosidases/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , beta-ManosidaseRESUMO
Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Retículo Endoplasmático/enzimologia , Metionina/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Fluoresceína-5-Isotiocianato , Fluoresceínas , Corantes Fluorescentes , Hidroxilação , Masculino , Octoxinol , Polietilenoglicóis , Ratos , Ratos Endogâmicos , Tensoativos , Tiocianatos , TripsinaRESUMO
The equilibria of the binding of methyl and ethyl isonitrile to carp hemoglobin have been measured at three pH values in the presence and absence of inositol hexaphosphate. The binding of methyl isonitrile is characterized by a higher overall dissociation constant, C1/2, and a higher Hill coefficient, n, than that of the ethyl derivative. The former is consistent with the greater hydrophobicity of ethyl isonitrile, and the latter is probably due to a greater intrinsic difference or heterogeneity in the binding affinities of the alpha- and beta-chains for the larger ligand. Changes in log C1/2 which result from alterations in pH or addition of organic phosphate are the same for both ligands within experimental error. This result is not consistent with affinity changes being the result of steric interactions between the protein and the ligand. At pH 6 in the presence of inositol hexaphosphate, equilibrium parameters estimated from overall rates of ligand binding and dissociation are in good agreement with direct equilibrium measurements. This is consistent with the protein being in a low-affinity, T-like state even when saturated with ligand under these conditions, resulting in a loss of cooperativity in ligand binding. At high pH, ligand binding remains cooperative, as evidenced by n values greater than unity, a general lack of agreement between measured equilibrium parameters and those estimated from overall kinetic constants, and differences in the kinetics of ligand binding as observed by rapid-mixing and flash photolysis techniques. Thus, the deoxygenated state of carp hemoglobin at high pH does not appear to be a good model of a deoxygenated R quaternary structural state.
Assuntos
Hemoglobinas/metabolismo , Nitrilas/metabolismo , Animais , Carpas , Concentração de Íons de Hidrogênio , Cinética , Ligantes/metabolismo , Ácido Fítico/metabolismo , Ligação ProteicaRESUMO
Phenobarbital-induced rat liver microsomes were subjected to NADPH- and Fe2+-catalyzed lipid peroxidation. The formation of approx. 95 nmol malondialdehyde/mg protein during 18 min peroxidation at 37 degrees C was observed. Membrane rigidity measured by means of the steady-state fluorescence anisotropy rs of diphenylhexatriene increased in parallel with the malondialdehyde formation. Both the amount of malondialdehyde and rs remained constant thereafter during incubation of the peroxidized membranes for 2 h. The aminopyrine demethylase activity decreased by about 60% upon lipid peroxidation for 18 min, whereas no significant loss of benzphetamine demethylase activity within the same time range was observed. A time-dependent formation of protein complexes of high molecular weight, comprising most of the microsomal polypeptides, upon lipid peroxidation was observed in SDS-polyacrylamide gel electrophoresis. The effect of microsomal lipid peroxidation on protein-protein interactions was examined by measuring the rotational mobility of intact cytochrome P-450. Rotational diffusion was measured by observing the decay of flash-induced absorption anisotropy r(t) of the P-450 X CO complex. Analysis was based on a 'rotation-about-membrane normal' model with the equation r(t) = r1exp(-t/phi 1) + r2exp(-t/phi 2). In control microsomes, two classes (rapid and slow) of rotating populations of cytochrome P-450 were observed with phi 1 approximately equal to 150 microseconds, fraction r1/(r1 + r2) approximately equal to 40% and phi 2 approximately equal to 2 ms, fraction r2/(r1 + r2) approximately equal to 60%. A relatively small decrease in the rotational mobility of P-450 was observed by a 18-min lipid peroxidation, while a subsequent incubation of peroxidized microsomes for 2 h at 37 degrees C resulted in a dramatic immobilization of P-450 by the increase of both r2/(r1 + r2) approximately equal to 75% and phi 2 approximately equal to 10-25 ms. The decrease in the P-450 mobility during 18-min lipid peroxidation would be due to the rigidification of the lipid bilayer. However, because the lipid fluidity remained unchanged thereafter, the significant immobilization of P-450 by the subsequent 2-h incubation is deduced to be due to formation of protein aggregates.
Assuntos
Sistema Enzimático do Citocromo P-450 , Peróxidos Lipídicos/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Eletroforese em Gel de Poliacrilamida , Polarização de Fluorescência , Masculino , Malondialdeído/biossíntese , Matemática , Fluidez de Membrana/efeitos dos fármacos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ratos , Ratos Endogâmicos , Temperatura , Fatores de TempoRESUMO
Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650-3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Microssomos Hepáticos/enzimologia , Alquilação , Animais , Membrana Celular/enzimologia , Cromatografia Líquida de Alta Pressão , Oxirredução , Peptídeos/química , Proteolipídeos , Ratos , Ratos Endogâmicos , TripsinaRESUMO
Cobalt hemoglobin Zürich (alpha 2 beta 263His leads to Arg) has been successfully reconstituted from the apohemoglobin Zürich and cobaltous protoporphyrin IX. The oxygen affinity of cobalt hemoglobin Zurich, as well as that of iron hemoglobin Zürich, were measured in the absence and presence of organic phosphate and Cl-. The overall oxygen affinity of cobalt hemoglobin Zürich was found to be higher and the cooperativity as measured by the n value was smaller than those of cobalt hemoglobin A. Organic phosphate and Cl- affect the oxygen equilibrium properties of cobalt hemoglobin Zürich in a manner similar to that of cobalt hemoglobin A, but to a lesser extant than cobalt hemoglobin A. The EPR spectrum of oxy cobalt hemoglobin Zürich is less sensitive to the replacement of the buffer system from H2O to 2H2O, indicating that the hydrogen bond between the distal amino acid residue and the bound oxygen is not formed in the abnormal beta subunits. The deoxy EPR spectrum of cobalt hemoglobin Zürich is similar to that of deoxy cobalt hemoglobin A, suggesting that the deoxy cobalt hemoglobin Zürich is predominantly in the deoxy quaternary structure (T state).
Assuntos
Cobalto/metabolismo , Hemoglobinas Anormais/metabolismo , Oxigênio/sangue , Fenômenos Químicos , Química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Oxirredução , Ligação ProteicaRESUMO
Both ATP and GTP are present in considerable amounts in red cells of the common goldfish Carassius auratus. They both influence the Root effect of the single major fish hemoglobin, but GTP is, depending on pH, 2-6 times more effective than ATP. The two triphosphates account for 3/4 of the effect of trichloroacetic acid supernatant obtained from hemolysate which contains still some compound(s) which can influence the shift of the Root effect toward higher pH.
Assuntos
Trifosfato de Adenosina/farmacologia , Eritrócitos/metabolismo , Guanosina Trifosfato/farmacologia , Hemoglobinas/metabolismo , Oxigênio , Fosfatase Alcalina/metabolismo , Animais , Carpa Dourada , Concentração de Íons de Hidrogênio , Ligação ProteicaRESUMO
The total methodology for a new mammalian erythroid cell system which permits direct investigation of the terminal stages of hemoglobin synthesis and assembly is described. The canine system allows quantitative separation of native heme containing alpha and beta chains which recombine to for tetrameric hemoglobin with normal functional properties (n = 2.17). These chains can be utilized for investigation in a cell-free system in which the rates of synthesis of alpha and beta chains are equal (alpha/beta = 0.96 +/- 0.05). Methodology for the quantitative separation of globin allows study of the effects of balanced and unbalanced globin chain synthesis in both the intact cells and the cell-free system.
Assuntos
Eritrócitos/metabolismo , Hemoglobinas/biossíntese , Modelos Biológicos , Animais , Cães , Globinas/biossíntese , Globinas/isolamento & purificação , Hemoglobinas/isolamento & purificação , Técnicas In Vitro , Precursores de Proteínas/biossíntese , Precursores de Proteínas/isolamento & purificaçãoRESUMO
The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an R-factor of 16.2 % utilizing synchrotron data in the resolution range from 10 to 1.7 A. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typical peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the LiP model an unprecedented modification of an amino acid was recognized. The surface residue tryptophan 171 in LiP is stereospecifically hydroxylated at the Cbeta atom due to an autocatalytic process. We propose that during the catalytic cycle of LiP a transient radical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-binding site centered at Trp171 has been reported, by us which is different from the "classical heme edge" site found in other peroxidases. Here, we report evidence for a radical formation at Trp171 using spin trapping, which supports the concept of Trp171 being a redox active amino acid and being involved in the oxidation of veratryl alcohol. On the basis of our current model, an electron pathway from Trp171 to the heme is envisaged, relevant for the oxidation of veratryl alcohol and possibly lignin. Beside the opening leading to the heme edge, which can accommodate small aromatic substrate molecules, a smaller channel giving access to the distal heme pocket was identified that is large enough for molecules such as hydrogen peroxide. Furthermore, it was found that in LiP the bond between the heme iron and the Nepsilon2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the heme more electron deficient and destabilizes high oxidation states, which could explain the higher redox potential of LiP as compared to CcP.
Assuntos
Peroxidases/química , Phanerochaete/enzimologia , Triptofano/química , Álcoois Benzílicos/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cristalografia por Raios X , Radicais Livres/metabolismo , Proteínas Fúngicas/química , Hemeproteínas/química , Ligação de Hidrogênio , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxirredução , Dobramento de Proteína , Estrutura Secundária de Proteína , Protoporfirinas/química , Espectrofotometria , Marcadores de SpinRESUMO
Structural features of the heme and the heme cavity of the monomeric hemoglobin (Hb) from the platyhelminth Dicrocoelium dendriticum were investigated by optical and proton nuclear magnetic resonance spectroscopy. Using nuclear Overhauser effects (NOEs) from resonances assigned previously through isotope labeling, most hyperfine-shifted resonances could be attributed to individual heme and protein protons in the cyano-metHb complex. It was observed that the heme 2-vinyl group is held in the trans orientation by nearby residues, whereas the 4-vinyl group exhibits an equilibrium between cis and trans orientations. NOE experiments in 1H2O allowed the identification of exchangeable protons belonging to the proximal histidine residue (F8) and to a distal residue. Detailed analysis of the NOE patterns obtained from the distal labile proton to non-labile protons and among these latter protons leads to the conclusion that a tyrosine side-chain occupies the distal site E7. Optical spectra of the alkaline-metHb also lead to this view, in that they are not typical of a hydroxy-metHb complex but instead resemble that of a hemin-phenolate or human mutant (M-type) Hb with a tyrosine residue linked to the iron atom. Further evidence for a distal tyrosine residue stems from the occurrence of an unusually stable transient ferrous Hb-cyanide complex, formed upon reduction of cyano-metHb to deoxy-Hb with dithionite. We suggest that the stability of this intermediate is due to a slow re-orientation of a large distal side-chain prior to cyanide dissociation. The sequence of the E-helix, known from the partially determined primary structure, was realigned to accommodate these findings. A frame-shift by one residue now positions a tyrosine at the distal site E7 instead of the originally proposed glycine residue.
Assuntos
Dicrocoelium , Heme , Aminoácidos , Animais , Hemoglobinas , Espectroscopia de Ressonância Magnética , Metemoglobina , Propionatos , Prótons , TirosinaRESUMO
The spectroscopic properties of Dicrocoelium dendriticum met-hemoglobin, investigated between pH 3.8 and 10.5, display two proton-induced transitions with apparent pK values of 8.1 and 4.7. The spectral changes, over the pH region 6.5 to 10.5, correspond to the high to low spin transition usually observed in ferric hemoproteins. The relaxation time (tau = 3.2 ms at the pK) associated with this transition is closely similar to that observed in Aplysia limacina met-myoglobin, but approximately 1000-fold slower than that of sperm whale met-myoglobin. The spectral changes associated with the more acid transition are in the opposite direction and have not been resolved by the temperature jump method. The rate constants for the reaction of azide with Dicrocoelium dendriticum met-hemoglobin were measured between pH 3.8 and 6.7 by the temperature jump method. Two kinetic schemes, both consistent with the pH dependence of the apparent rate constant for binding of azide, were identified. No objective way of discriminating between the two models is available. However, one of them is amenable to a physical interpretation based on a comparison with the structural, kinetic and spectral properties of other monomeric hemoproteins.
Assuntos
Azidas/metabolismo , Dicrocoelium , Metemoglobina/metabolismo , Animais , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Ligação Proteica , EspectrofotometriaRESUMO
The 1H nuclear magnetic resonance characteristics of met-cyano and met-aquo hemoglobin from the sheep bile duct parasite Dicrocoelium dendriticum have been compared to those of other monomeric hemoglobins and myoglobins. By varying temperature and pH, it was found that the studied material is a mixture of several isozymes differing slightly in their structural features around the heme cavity. The heme in-plane rhombic asymmetry, as indicated by the spread of the heme methyl hyperfine shifts, is intermediate between that of sperm whale myoglobin and leghemoglobin. The proximal histidine is present and its dynamic properties, as probed by the exchange of the ring NH with bulk solvent protons, point towards a cavity more stable than those of sperm whale myoglobin and leghemoglobin. In the met-cyano form, an exchangeable proton was detected close to the iron center that was tentatively assigned to an arginine residue located three amino acid residues closer to the C terminus than the proximal histidine. The transition from the met-aquo form to the met-hydroxy form occurring at pH 8.1 and previously detected by optical methods was observed. Furthermore, consideration of the mean heme methyl hyperfine shift average indicates that the iron remains six-co-ordinate down to below pH 4.5 irrespective of an acid-transition (pK approximately 5) in the protein. However, the presence of a "pseudo" six-co-ordinate (i.e. high-spin, in-plane, five-co-ordinate) iron at pH values below the acid-transition pK cannot be excluded on the basis of the presently available data. The pH dependence of several resonances in both the met-cyano and met-aquo forms of the protein reflect a pK value compatible with the titration of a heme propionate.