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1.
Biochim Biophys Acta ; 924(1): 67-74, 1987 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-2435326

RESUMO

We used a gel filtration method and a stable prostaglandin I2 (prostacyclin) analogue, iloprost, to study the kinetics of prostaglandin I2 binding by human serum proteins. Binding equilibrium experiments conducted at physiological prostaglandin I2 concentration (nM) yielded a KD of 10(-9) and a capacity of approx. 50 nM for the serum binding protein(s). Kinetic measurements gave a dissociation rate constant of 10(-3) s-1. When binding equilibrium was established at various ligand concentrations ranging from nM to microM, a result indicating an unsaturable binding was obtained utilizing this method. On the other hand, saturation was achieved with a ligand concentration as high as 50-100 microM by another binding method. A KD of 7 X 10(-5) and a capacity of approx. 600 microM was obtained. This apparent discrepancy was resolved by performing parallel experiments using purified human serum albumin samples and serum. It is concluded that the large quantity of serum albumin, approx. 600 microM, in serum may compensate for its low KD (approx. 10(-5] for prostaglandin I2, thus simulating a binding protein with a KD of 10(-9) and a limited capacity. These data offer direct information regarding how prostaglandin I2 is stabilized by serum and is transported to the platelet prostaglandin I2 receptors. There is a strong implication that serum albumin is the major if not the only protein responsible for binding of prostaglandin I2.


Assuntos
Proteínas Sanguíneas/metabolismo , Epoprostenol/sangue , Proteínas Sanguíneas/isolamento & purificação , Humanos , Iloprosta , Cinética , Ligação Proteica
2.
Biochim Biophys Acta ; 852(1): 100-5, 1986 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-3021213

RESUMO

Treatment of yeast Complex III with hexane or repeated fractionation with ammonium sulfate-cholate abolishes the ability of antimycin to bind to the complex. Antimycin binding is partially restored by addition of phospholipids to the depleted complex; this action of phospholipids can be enhanced by including Q6 in the reconstitution mixture.


Assuntos
Antimicina A/análogos & derivados , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fosfolipídeos/farmacologia , Antimicina A/metabolismo , Cardiolipinas/farmacologia , Fluorescência , Ubiquinona/fisiologia
3.
Biochim Biophys Acta ; 942(2): 220-6, 1988 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-2456096

RESUMO

Iloprost, a stable analog of prostacyclin, has been used for studying the interaction between prostacyclin and its effector cells such as platelets and vascular cells. The compound is usually prepared as a mixture of 16(S) and 16(R) stereoisomers. In this work, we compared the biological activity and platelet receptor binding characteristics between the two isomers. The 16(S) isomer was 20-times more potent than the 16(R) in inhibiting collagen-induced platelet aggregation. Equilibrium binding of iloprost isomers to platelet membrane receptors measured by rapid filtration method revealed that the specific binding data of 16(S) isomer was fit for a single binding species with Kd of 13.4 nM and Bmax 665 fmol/mg protein. By contrast, the Kd and Bmax of 16(R) isomer were 288 nM and 425 fmol/mg, respectively. To further assess different binding behavior of these two isomers, association rate was measured. The observed association rate of the S isomer was 0.036 s-1 and 0.001 s-1 for the R isomer at 15 nM iloprost and 2 mg/ml platelet membrane proteins. We postulate that the striking difference in the association rate with resultant difference in binding affinity and biologic activity between the two isomers was due to fitting of the molecule to the receptor channel. The 16(S) form has a more favorable orientation for fitting into the receptor. We conclude that the two iloprost isomers must be considered as two entirely different compounds when iloprost is used as the ligand for quantifying prostacyclin receptor binding.


Assuntos
Plaquetas/metabolismo , Epoprostenol/sangue , Receptores de Prostaglandina/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Iloprosta , Isomerismo , Cinética , Modelos Moleculares , Receptores de Epoprostenol
4.
Biochim Biophys Acta ; 806(3): 418-26, 1985 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-2982396

RESUMO

We have examined the effects of eight inhibitors of the bovine-heart mitochondrial Complex III on the catalytic activity of the analogous complex from yeast mitochondria. All eight compounds were inhibitory, with potent inhibition being obtained with antimycin, myxothiazol and UHDBT (5-N-undecyl-6-hydroxy-4,7-dioxobenzothiazole). These three inhibitors, and also funiculosin, have been further studied by characterizing their effects on the visible absorbance, magnetic circular dichroism and EPR spectra of the complex and also on the potentiometric properties of the individual metal centers present in the complex. All four inhibitors had little or no effect on either the absorbance or magnetic circular dichroism spectra. Funiculosin produced a change in the EPR lineshape of the iron-sulfur cluster; EPR spectra recorded at 12 K also revealed complete reduction of cytochrome b-562 by ascorbate. UHDBT also changed the lineshape of the iron-sulfur cluster and this change could be partially reversed by myxothiazol. Neither antimycin nor myxothiazol affected the iron-sulfur cluster and produced only small changes in the EPR absorption envelope of the b cytochromes. Both funiculosin and UHDBT raised the midpoint potential of the iron-sulfur cluster, by about 150 and 70 mV, respectively. Only UHDBT changed the potential of c1, lowering it by about 30 mV. Funiculosin raised the potential of b-562 by about 30 mV, while myxothiazol had no effect; the other two compounds produced only small changes. All four compounds had only small effects on the midpoint potential of b-566. The relative contributions of the two b cytochromes to the magnetic circular dichroism amplitudes could be changed by the addition of inhibitors, even though the absolute magnetic circular dichroism spectra of oxidized and reduced complex were unaffected.


Assuntos
Complexos Multienzimáticos/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , Quinona Redutases/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Antraquinonas/farmacologia , Antimicina A/farmacologia , Dicroísmo Circular , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Complexo III da Cadeia de Transporte de Elétrons , Proteínas Ferro-Enxofre/metabolismo , Metacrilatos , Oxirredução , Espectrofotometria , Tiazóis/farmacologia
5.
Biochim Biophys Acta ; 1115(2): 131-40, 1991 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-1764464

RESUMO

Although previous studies have shown that serum albumin binds PGI2 and protects it from rapid degradation, it remains debatable whether it is physiologically important due to its low binding affinity for PGI2. We were intrigued by the observations of Yui et al. (J. Clin. Invest. 82 (1988) 803-807) which suggested that apo A-I of the high density lipoprotein (HDL) is the "serum PGI2 stabilizing factor". To clarify this, we carried out experiments to determine the binding kinetics and parameters of HDL and albumin purified from normal pooled human serum. Despite the use of multiple binding assays, we could not detect any binding activity in HDL2, HDL3 or nascent HDL preparations, nor could we demonstrate any PGI2 protecting activity by these molecules. By contrast, purified albumin exhibited essentially identical binding parameters as the native serum from which the albumin was purified. The binding activity of various albumin preparations was not due to the contamination of apo A-I. Computer simulation analysis also failed to provide evidence to support the notion that HDL bound and prolonged PGI2 activity. To determine whether physiological concentrations of albumin influence PGI2 binding to platelet receptors, we measured PGI2 binding to platelet membrane in the absence and presence of albumin. Albumin at 40 mg/ml increased the KD of PGI2 binding to the receptors by 2-3 fold. These findings indicate that albumin plays a major role in protecting PGI2 activity and regulating its availability for platelet PGI2 receptors.


Assuntos
Epoprostenol/sangue , Lipoproteínas HDL/sangue , Albumina Sérica/metabolismo , Apolipoproteína A-I/metabolismo , Sítios de Ligação , Disponibilidade Biológica , Plaquetas/metabolismo , Membrana Celular/metabolismo , Meia-Vida , Humanos , Receptores de Epoprostenol , Receptores de Prostaglandina/metabolismo , Albumina Sérica/isolamento & purificação
6.
Biochim Biophys Acta ; 993(1): 74-82, 1989 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-2478194

RESUMO

We synthesized a fluorescent probe, 2,6-dichloro-4-aminophenol iloprost or dichlorohydroxyphenylamide of iloprost (DCHPA-iloprost) by reacting the stable prostacyclin analog, iloprost (ZK 35 374), with 2,6-dichloro-4-aminophenol with a yield of 60%. This probe exhibited an optical spectrum which overlapped with the emission spectrum of the sole tryptophan of human serum albumin (HSA). Energy transfer from the tryptophan residue to the phenol moiety of DCHPA-iloprost was observed. We utilized this donor-quenching phenomenon to quantitate the binding stoichiometry and affinity as well as the association rate of DCHPA-iloprost binding to HSA. As DCHPA-iloprost showed similar binding characteristics similar to those of iloprost and prostacyclin and competed with iloprost for HSA binding sites, we used DCHPA-iloprost as a probe to locate the binding domain of prostacyclin (PGI2) in HSA. The distance between the tryptophan indole and the phenol group of DCHPA-iloprost was estimated to be 15-18 A. Because iloprost binding to HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA molecules. A possible molecular mechanism by which HSA reduces the chemical degradation of PGI2 and stabilizes its activity could be derived from this model.


Assuntos
Epoprostenol/sangue , Corantes Fluorescentes/síntese química , Albumina Sérica/metabolismo , Sítios de Ligação , Transferência de Energia , Humanos , Iloprosta , Indicadores e Reagentes , Espectrometria de Fluorescência
7.
Biochim Biophys Acta ; 848(3): 305-11, 1986 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-3004577

RESUMO

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids.


Assuntos
Azidas/metabolismo , Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Ubiquinona/análogos & derivados , Marcadores de Afinidade , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons , Complexos Multienzimáticos/metabolismo , Fosfolipídeos/metabolismo , Fotólise , Quinona Redutases/metabolismo , Succinatos/metabolismo , Ubiquinona/metabolismo
8.
Protein Sci ; 7(12): 2550-9, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9865949

RESUMO

Stringent specificity and complementarity between the receptor, a periplasmic phosphate-binding protein (PBP) with a two-domain structure, and the completely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site is intensely electronegative. This novel finding prompted the study reported here of the effect of ionic strength on the equilibrium and rapid kinetics of phosphate binding. To facilitate this study, Ala197, located on the edge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fluorescence reporter group. The A197W PBP-phosphate complex retains wild-type Kd and X-ray structure beyond the replacement residue. The Kd (0.18 microM) at no salt is increased by 20-fold at greater than 0.30 M NaCl. Stopped-flow fluorescence kinetic studies indicate a two-step binding process: (1) The phosphate (L) binds, at near diffusion-controlled rate, to the open cleft form (Po) of PBP to produce an intermediate, PoL. This rate decreases with increasing ionic strength. (2) The intermediate isomerizes to the closed-conformation form, PcL. The results indicate that the high specificity, affinity, and rate of phosphate binding are not influenced by the noncomplementary electronegative surface potential of the cleft. That binding depends almost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in ligand recognition.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fosfatos/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Cristalografia por Raios X , Fluorescência , Cinética , Modelos Moleculares , Mutação , Concentração Osmolar , Proteínas de Ligação a Fosfato , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Propriedades de Superfície , Triptofano
9.
FEBS Lett ; 214(1): 117-21, 1987 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-3032676

RESUMO

Yeast mitochondrial complex III contains a subunit with a [2Fe-2S] cluster (the Rieske center) that has unusual physical and chemical properties. For apparently similar centers isolated from bacteria, it has been shown by electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) measurements that these [2Fe-2S] centers are coordinated by at least one and probably two nitrogen ligands. This work describes similar ENDOR and ESEEM studies on the intact mitochondrial complex. We find that this [2Fe-2S] cluster exhibits ESEEM and ENDOR properties that appear to be indistinguishable from those observed with the isolated bacterial systems. Furthermore, changes in EPR lineshape that occur as complex III is progressively reduced are not accompanied by any changes in the nitrogen coupling parameters. This spectroscopic evidence for nitrogen coordination is supported by published sequence data on four Rieske iron-sulfur subunits. It seems likely that this is a general characteristic of such [2Fe-2S] redox active centers.


Assuntos
Proteínas de Bactérias , Complexo III da Cadeia de Transporte de Elétrons , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Ferro , Espectroscopia de Ressonância Magnética , Nitrogênio , Oxirredução
10.
Thromb Res ; 51(6): 583-92, 1988 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-3055418

RESUMO

We quantitated serum PGI2 binding of 8 normal subjects and two TTP (thrombotic thrombocytopenic purpura) patients by gel filtration and gel partition methods using a stable PGI2 analogue, iloprost. The dissociation constant (KD) and the binding capacity (or binding stoichiometry) determined for the normals were 94 +/- S.D. 19 microM and 1.8 +/- S.D. 0.5 mM (or 2.0 +/- .6, iloprost:HSA). Corresponding values for serum samples obtained from TTP patient I were KD 200 microM, and Bmax 2.3 mM in the acute phase, and 75 microM and 1.8 mM respectively in the remission phase. The serum samples from TTP patient II exhibited a higher KD. Values of 299 microM (acute phase) and 147 microM (remission phase) were obtained. The corresponding binding capacities were 2.1 mM and 1.5 mM. Binding affinity change appears to be the main factor which resulted in the PGI2 binding defect in TTP.


Assuntos
Epoprostenol/sangue , Trombocitopenia/sangue , Adulto , Ligação Competitiva , Feminino , Humanos , Masculino
11.
Acta Anaesthesiol Sin ; 35(3): 133-9, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9407676

RESUMO

BACKGROUND: Severe hemorrhagic tendency often complicates cardiopulmonary bypass (CPB) in cardiac surgery. In this study, we compared the effectiveness of thromboelastography (TEG), Sonoclot (SCT), and routine coagulation test (RCT) in the prediction of coagulation defects. METHODS: Forty-three patients undergoing cardiac surgery with CPB were included. Blood for RCT, TEG, and SCT profiles was sampled before systemic heparinization and after protamine administration. Clinically significant bleeding was defined as chest tube drainage in excess of 100 ml/h for 3 consecutive hours or 300 ml/h in 1 h. All coagulation parameters obtained before and after CPB were compared. The sensitivity, specificity, accuracy, false positive, and false negative rate were also calculated and compared. RESULTS: All coagulation tests were within normal range except higher partial thromboplastin time. Variables which were significantly different from those before CPB included platelet count, fibrinogen level, prothrombin time, and thrombin time in RCT, alpha angle and maximum amplitude in TEG, and R2 and peak time in SCT. In the TEG tracing, all variables had high sensitivity, specificity, and accuracy (average 85.4%, 83%, and 83.5% respectively) and low false positive and negative rate (12.5% and 5% respectively). Although SCT had high sensitivity (76.3%) and low false negative rate (6.5%), its specificity and accuracy were all under 50%. CONCLUSIONS: Our data demonstrated that the TEG monitoring is a useful tool for detecting post-CPB bleeding diathesis and can provide much predictive information. RCT and SCT are of limited value because of higher rate of unreliable results.


Assuntos
Testes de Coagulação Sanguínea , Procedimentos Cirúrgicos Cardíacos , Ponte Cardiopulmonar , Transtornos Hemorrágicos/diagnóstico , Tromboelastografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Biochemistry ; 39(31): 9373-83, 2000 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-10924132

RESUMO

Endothelial nitric oxide synthase (eNOS) is a self-sufficient P450-like enzyme. A P450 reductase domain is tethered to an oxygenase domain containing the heme, the substrate (L-arginine) binding site, and a cofactor, tetrahydrobiopterin (BH(4)). This "triad", located at the distal heme pocket, is the center of oxygen activation and enzyme catalysis. To probe the relationships among these three components, we examined the binding kinetics of three different small heme ligands in the presence and absence of either L-arginine, BH(4), or both. Imidazole binding was strictly competitive with L-arginine, indicating a domain overlap. BH(4) had no obvious effect on imidazole binding but slightly increased the k(on) for L-arginine. L-Arginine decreased the k(on) and k(off) for cyanide by two orders, indicating a "kinetic obstruction" mechanism. BH(4) slightly enhanced cyanide binding. Nitric oxide (NO) binding kinetics were more complex. Increasing the L-arginine concentration decreased the NO binding affinity at equilibrium. In both BH(4)-abundant and BH(4)-deficient eNOS, half of the NO binding sites showed a sizable decrease of the binding rate by L-arginine, with the rate of NO binding at the other half of the sites remaining essentially unaltered by L-arginine, implying that the two heme centers in the eNOS dimer are functionally distinct.


Assuntos
Arginina/química , Biopterinas/química , Cianetos/química , Compostos Férricos/química , Heme/química , Imidazóis/química , Óxido Nítrico Sintase/química , Óxido Nítrico/química , Animais , Ligação Competitiva , Biopterinas/análogos & derivados , Catálise , Bovinos , Simulação por Computador , Escherichia coli/genética , Flavinas/química , Vetores Genéticos/síntese química , Humanos , Ligantes , Modelos Químicos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/isolamento & purificação , Óxido Nítrico Sintase Tipo III , Ligação Proteica
13.
Eicosanoids ; 2(3): 131-43, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2517029

RESUMO

Correlation analysis between the structural changes of PGI2 and the corresponding changes in platelet activity was performed to look into the following structural features: (1) the spatial relationship of critical functional groups, (2) conformational flexibility of the molecule as a result of chemical modification; (3) charge distribution around C6a; (4) hydrogen-bonding capability and mispairing and (5) the steric effects which resulted from chemical modifications. Our studies led to three important conclusions: (1) The C1 carboxylate and C11, C15 hydroxyl groups of PGI2 are essential for platelet activity. Modifications that change their relative positions reduce the activity. The ring structure and the C5 and C13 double bonds are molecular designs to maintain this unique geometry. (2) The C6a oxygen, although not vital to the binding geometry, is important for the biological potency. Decreasing the electronegativity of C6a oxygen leads to decreased potency. (3) We propose that any additional hydrogen-bond donor present between C1 and C15 could cause a hydrogen-bond mispairing and therefore a decreased activity. These findings should be useful for designing new PGI2 analogues and for determining the configuration of receptor-associated PGI2 by a molecular mechanics technique.


Assuntos
Plaquetas/efeitos dos fármacos , Epoprostenol/farmacologia , Receptores de Prostaglandina/efeitos dos fármacos , Animais , Plaquetas/metabolismo , Eletroquímica , Epoprostenol/metabolismo , Ligação de Hidrogênio , Técnicas In Vitro , Estrutura Molecular , Prostaglandinas Sintéticas/metabolismo , Prostaglandinas Sintéticas/farmacologia , Receptores de Epoprostenol , Receptores de Prostaglandina/metabolismo , Relação Estrutura-Atividade
14.
Arch Biochem Biophys ; 383(1): 119-27, 2000 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-11097184

RESUMO

Thromboxane A2 synthase (TXAS) is a member of the cytochrome P450 superfamily and catalyzes an isomerization reaction that converts prostaglandin H2 to thromboxane A2. As a step toward understanding the structure/function relationships of TXAS, we mutated amino acid residues predicted to bind the propionate groups of A- and D-pyrrole rings of the heme. These mutations at each of these residues (Asn-110, Trp-133, Arg-137, Arg-413, and Arg-478) resulted in altered heme binding, as evidenced by perturbation of the absorption spectra and EPR. The mutations, although causing no significant changes in the secondary structure of the proteins, induced tertiary structural changes that led to increased susceptibility to trypsin digestion and alteration of the intrinsic protein fluorescence. Moreover, these mutant proteins lost their binding affinity to the substrate analog, had a lower heme content and retained less than 5% of the wild-type catalytic activity. However, mutations at the neighboring amino acid of the aforementioned residues yielded mutant proteins retaining the biochemical and biophysical properties of the wild type TXAS. Aligning the TXAS sequence with the structurally known P450s, we proposed that in TXAS the A-ring propionate of the heme is hydrogen bonded to Asn-110, Arg-413, and Arg-478, whereas D-ring propionate is hydrogen bonded to Trp-133 and Arg-137. Furthermore, both A- and D-ring propionates bulge away from the heme plane and both lie on the proximal face of heme plane, a structure similar to P450terp.


Assuntos
Heme/metabolismo , Tromboxano-A Sintase/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Dados de Sequência Molecular , Mutação , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato , Tromboxano-A Sintase/genética
15.
Arch Biochem Biophys ; 313(2): 367-72, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8080285

RESUMO

Prostaglandin H synthase (PGHS) is a hemeprotein, and thus its catalytic activity potentially could be modulated by direct interaction with nitric oxide (NO). We have monitored spectroscopic and activity changes in pure ovine PGHS isoform-1 to investigate its interaction with NO in more detail. The binding kinetics for NO and the ferric heme in resting PGHS were analyzed by stopped-flow spectrophotometry at 21 degrees C. The rate constants for association and dissociation were estimated to be 6.5 x 10(4) M-1 s-1 and 60 s-1, respectively, leading to an equilibrium dissociation constant (Kd) of 0.92 mM. NO thus has a relatively weak affinity for heme in ferric PGHS, the resting oxidation state of this hemeprotein. NO did react strongly and completely with ferrous PGHS under anaerobic conditions, displacing the proximal histidine ligand to the prosthetic group. Dissolved NO at up to 2 mM produced only slight decreases in the cyclooxygenase activity of microsomal, detergent-extracted, or homogeneous preparations of ovine PGHS. The NO donors sodium nitroprusside and glyceryl trinitrate at levels of up to 1 mM also had little effect on the activity of the PGHS preparations. Thus, there was no evidence for significant direct interaction of PGHS with NO at concentrations likely to be encountered in vivo.


Assuntos
Óxido Nítrico/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Heme/metabolismo , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Masculino , Óxido Nítrico/farmacologia , Nitroglicerina/farmacologia , Nitroprussiato/farmacologia , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/isolamento & purificação , Glândulas Seminais/enzimologia , Ovinos , Espectrofotometria
16.
J Comput Aided Mol Des ; 5(2): 135-48, 1991 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1869897

RESUMO

To elucidate the conformation of receptor-associated prostacyclin (PGI2), we first performed structure-activity correlation analysis of over 200 PGI2 analogues and derived from this analysis several crucial features pertaining to structural requirements for PGI2 activity [Ah-lim Tsai and Kenneth K. Wu, Eicosanoids, 2 (1989) 131-143]. These structural features proved to be useful guidelines for selecting 'model molecules' for further investigations by molecular mechanics. By properly selecting four analogues with either rigid or uniquely oriented alpha-side chain structure for geometric fitting, we succeeded in maximally minimizing the degree of freedom of the carboxylate terminus of PGI2. We were able to define the spatial relationship among the four critical functional groups, i.e., C1-COOH, C6a-O, C11-OH and C15-OH. More information is needed, however, to define the geometry of the omega-side chain, particularly for the moiety beyond C15. Nevertheless, results from structure-activity correlation analysis and molecular modeling provide useful information regarding the conformation of receptor-associated PGI2, which assumes an 'elongated' conformation instead of the traditional 'hairpin' structure.


Assuntos
Simulação por Computador , Epoprostenol/análogos & derivados , Modelos Moleculares , Sítios de Ligação/fisiologia , Conformação Molecular , Software , Relação Estrutura-Atividade
17.
J Biol Chem ; 267(25): 17753-9, 1992 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-1325448

RESUMO

Hydroperoxides are known to induce the formation of tyrosyl free radicals in prostaglandin (PG) H synthase. To evaluate the role of these radicals in cyclooxygenase catalysis we have analyzed the temporal correlation between radical formation and substrate conversion during reaction of the synthase with arachidonic acid. PGH synthase reacted with equimolar levels of arachidonic acid generated sequentially the wide doublet (34 G peak-to-trough) and wide singlet (32 G peak-to-trough) tyrosyl radical signals previously reported for reaction with hydroperoxide. The kinetics of formation and decay of the doublet signal corresponded reasonably well with those of cyclooxygenase activity. However, the wide singlet free radical signal accumulated only after prostaglandin formation had ceased, indicating that the wide singlet is not likely to be an intermediate in cyclooxygenase catalysis. When PGH synthase was reacted with 25 equivalents of arachidonic acid, the wide doublet and wide singlet radical signals were not observed. Instead, a narrower singlet (24 G peak-to-trough) tyrosyl radical was generated, similar to that found upon reaction of indomethacin-treated synthase with hydroperoxide. Only about 11 mol of prostaglandin were formed per mol of synthase before complete self-inactivation of the cyclooxygenase, far less than the 170 mol/mol synthase produced under standard assay conditions. Phenol (0.5 mM) increased the extent of cyclooxygenase reaction by only about 50%, in contrast to the 460% stimulation seen under standard assay conditions. These results indicate that the narrow singlet tyrosyl radical observed in the reaction with high levels of arachidonate in this study and by Lassmann et al. (Lassmann, G., Odenwaller, R., Curtis, J.F., DeGray, J.A., Mason, R.P., Marnett, L.J., and Eling, T.E. (1991) J. Biol. Chem. 266, 20045-20055) is associated with abnormal cyclooxygenase activity and is probably nonphysiological. In titrations of the synthase with arachidonate or with hydroperoxide, the loss of enzyme activity and destruction of heme were linear functions of the amount of titrant added. Complete inactivation of cyclooxygenase activity was found at about 10 mol of arachidonate, ethyl hydrogen peroxide, or hydrogen peroxide per mol of synthase heme; maximal bleaching of the heme Soret absorbance peak was found with 10 mol of ethyl hydroperoxide or 20 mol of either arachidonate or hydrogen peroxide per mol of synthase heme. The peak concentration of the wide doublet tyrosyl radical did not change appreciably with increased levels of ethyl hydroperoxide. In contrast, higher levels of hydroperoxide gave higher levels of the wide singlet radical species, in parallel with enzyme inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Prostaglandina-Endoperóxido Sintases/metabolismo , Tirosina , Animais , Ácido Araquidônico/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Heme/metabolismo , Cinética , Peróxidos Lipídicos/metabolismo , Masculino , Microssomos/enzimologia , Glândulas Seminais/enzimologia , Ovinos , Fatores de Tempo
18.
J Biol Chem ; 262(22): 10524-31, 1987 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-3038886

RESUMO

We have examined the optical, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectra of pure ovine prostaglandin H synthase in its resting (ferric) and ferrous states and after addition of hydrogen peroxide or 15-hydroperoxyeicosatetraenoic acid. In resting synthase, the distribution of heme between high- and low-spin forms was temperature-dependent: 20% of the heme was low-spin at room temperature whereas 50% was low-spin at 12 K. Two histidine residues were coordinated to the heme iron in the low-spin species. Anaerobic reduction of the synthase with dithionite produced a high-spin ferrous species that had no EPR signals. Upon reaction with the resting synthase, both hydroperoxides quickly generated intense (20-40% of the synthase heme) and complex EPR signals around g = 2 that were accompanied by corresponding decreases in the intensity of the signals from ferric heme at g = 3 and g = 6. The signal generated by HOOH had a doublet at g = 2.003, split by 22 G, superimposed on a broad component with a peak at g = 2.085 and a trough at g = 1.95. The lipid hydroperoxide generated a singlet at g = 2.003, with a linewidth of 25 G, superimposed on a broad background with a peak at g = 2.095 and a trough around g = 1.9. These EPR signals induced by hydroperoxide may reflect synthase heme in the ferryl state complexed with a free radical derived from hydroperoxide or fragments of hydroperoxide.


Assuntos
Heme , Peróxido de Hidrogênio/farmacologia , Leucotrienos , Prostaglandina-Endoperóxido Sintases , Animais , Ácidos Araquidônicos/farmacologia , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Férricos , Compostos Ferrosos , Radicais Livres , Peróxidos Lipídicos/farmacologia , Masculino , Oxirredução , Glândulas Seminais/enzimologia , Ovinos , Espectrofotometria
19.
J Lipid Mediat ; 6(1-3): 145-54, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8395237

RESUMO

Reaction of ovine PGH synthase with arachidonic acid or hydroperoxides produces several tyrosine radical species that can be distinguished by electron paramagnetic resonance (EPR) spectroscopy. We have correlated the temporal sequence of the EPR signals with optical changes of the heme center, and with product formation. The synthase was reconstituted with either heme (Fe-PGHS) or Mn protoporphyrin IX (Mn-PGHS). Incubation of Fe-PGHS with equimolar arachidonate resulted in rapid appearance of a wide doublet tyrosyl radical EPR signal (34 G peak-to-trough); the intensity was near maximal by 7 s. The doublet gave way over the next 10 s to a wide singlet (32 G peak-to-trough) which peaked at 46 s and then decayed slowly. Electronic absorbance spectra indicated that formation of peroxidase Compound I was complete within 1 s; accumulation of peroxidase Compound II paralleled accumulation of the wide doublet tyrosyl radical. PGG2 and PGH2 accumulated rapidly during the first 5 s of reaction; little arachidonate remained after 12 s. The tyrosyl radical giving the wide doublet EPR signal is thus the best candidate for the oxidizing species postulated to abstract the 13S hydrogen atom from arachidonate during cyclooxygenase catalysis by Fe-PGHS. Incubation of Mn-PGHS with arachidonate also led to rapid generation of an oxidized peroxidase cycle intermediate, a protein-linked free radical, and prostaglandins. The radical signal seen with Mn-PGHS (singlet, 36 G peak-to-trough) was distinct from those observed with Fe-PGHS, but the kinetics of the Mn-PGHS radical were consistent with participation in cyclooxygenase catalysis.


Assuntos
Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Ácido Araquidônico/metabolismo , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/metabolismo , Peróxido de Hidrogênio/metabolismo , Técnicas In Vitro , Ferro/metabolismo , Cinética , Manganês/metabolismo , Ovinos , Especificidade por Substrato
20.
Biochemistry ; 34(26): 8499-512, 1995 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-7599139

RESUMO

Two types of mechanisms have been proposed to account for the combination of peroxidase and cyclooxygenase activities in prostaglandin H synthase (PGHS). One, a branched-chain mechanism [Dietz, R., et al. (1988) Eur. J. Biochem. 171, 321-328], postulates that the cyclooxygenase reaction propagates essentially independently of peroxidase catalysis. The second, a tightly coupled mechanism [Bakovic, M., & Dunford, H. B. (1994) Biochemistry 33, 6475-6482], postulates that peroxidase catalysis is an integral part of cyclooxygenase propagation. Qualitative and quantitative predictions from the two mechanisms have been compared with several observed characteristics of the PGHS reaction with arachidonate, including the ability to accumulate PGG2 and oxidized enzyme intermediates, the stoichiometry between cosubstrate and fatty acid consumption, and the hydroperoxide activator requirement. The observed characteristics, particularly the accumulation of micromolar levels of PGG2 even in the presence of cosubstrate and the stoichiometry between cosubstrate oxidation and fatty acid oxygenation of less than 1.3 (compared to a theoretical maximum of 2.0), were largely consistent with predictions from the branched-chain mechanism, but contradicted important predictions of the tightly coupled mechanism. These results indicate that PGHS catalysis is more accurately described by the branched-chain mechanism than by the tightly coupled mechanism.


Assuntos
Modelos Teóricos , Peroxidases/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Ácidos Araquidônicos/isolamento & purificação , Ácidos Araquidônicos/metabolismo , Cromatografia em Camada Fina , Ácidos Graxos/metabolismo , Cinética , Masculino , Oxirredução , Peroxidases/química , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/isolamento & purificação , Glândulas Seminais/enzimologia , Ovinos
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