RESUMEN
The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.
Asunto(s)
Flavina-Adenina Dinucleótido/deficiencia , Enfermedad Granulomatosa Crónica/enzimología , NADH NADPH Oxidorreductasas/sangre , NADPH Oxidasas , NADP/farmacología , Neutrófilos/enzimología , Complejo de Proteína del Fotosistema II , Adolescente , Adulto , Grupo Citocromo b/sangre , Flavina-Adenina Dinucleótido/farmacología , Humanos , Lactante , Masculino , Oxidación-Reducción , Espectrometría de Fluorescencia , Fracciones Subcelulares/enzimologíaRESUMEN
Knowledge of the sequence of a bioactive protein (angiotensinogen) and the availability of a natural product inhibitor lead (pepstatin) were the starting point for discovery of potent penta- and hexapeptide renin inhibitors. Study of the metabolism and disposition of these substances forced the discovery of simpler inhibitors leading to the discovery of oral activity in Terlakiren (22). Modification of physical properties led to the synthesis of aminopiperidine 30, which was identified by oral efficacy profiling. Structural modification to give enzymatic stability produced the bioavailable benzylsuccinate inhibitor 34. Its bioactive monomethylamine metabolite (35, CP-108,671) was subsequently found to have uniformly high oral bioavailability and activity in various species including primates.
Asunto(s)
Oligopéptidos/farmacología , Oligopéptidos/farmacocinética , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/farmacocinética , Renina/antagonistas & inhibidores , Renina/química , Administración Oral , Secuencia de Aminoácidos , Aminocaproatos/administración & dosificación , Aminocaproatos/farmacocinética , Aminocaproatos/farmacología , Animales , Sitios de Unión , Disponibilidad Biológica , Presión Sanguínea/efectos de los fármacos , Quimotripsina/antagonistas & inhibidores , Cobayas , Humanos , Cinética , Datos de Secuencia Molecular , Oligopéptidos/administración & dosificación , Inhibidores de Proteasas/administración & dosificación , Conformación Proteica , Renina/sangre , Solubilidad , Relación Estructura-ActividadRESUMEN
Chronic granulomatous disease (CGD) is a genetically transmitted disorder thought to result from defect(s) in the activation or turnover of the NADPH dependent O2- generating oxidase enzyme system of human neutrophils and monocytes. The normal oxidase may be a flavoprotein-cytochrome b559 complex; therefore, these components of the oxidase were quantitated in the neutrophils from patients and family members of two unrelated CGD kindreds. The male propositus from an X-linked recessive kindred had a neutrophil oxidase fraction with low FAD content (26 pmol/mg protein) and undetectable cytochrome b559 (less than 5 pmol/mg protein). The male propositus from an autosomal recessive kindred had a neutrophil oxidase fraction with low FAD content (34 pmol FAD/mg protein), but normal cytochrome b559 content (170 pmol cytochrome b559/mg protein). Both parents of this latter CGD patient had normal FAD and cytochrome b559 content in their neutrophil oxidase fraction. We conclude that the carrier state in certain X-linked recessive female carriers of CGD can be detected by partial deficiencies of both flavoprotein and cytochrome b559 components of the oxidase, whereas presumed heterozygous carriers of certain autosomal recessive CGD kindreds cannot be detected by this means.
Asunto(s)
Enfermedad Granulomatosa Crónica/metabolismo , Neutrófilos/metabolismo , Fagocitos/metabolismo , Complejo de Proteína del Fotosistema II , Superóxidos/metabolismo , Niño , Grupo Citocromo b/análisis , Femenino , Flavina-Adenina Dinucleótido/análisis , Radicales Libres , Enfermedad Granulomatosa Crónica/genética , Heterocigoto , Humanos , Masculino , Nitroazul de Tetrazolio/metabolismoRESUMEN
The enzymatic activity underlying the respiratory burst in human neutrophils was examined in a subcellular fraction with high specific activity and shown to be a membrane-associated complex of a flavoprotein, ubiquinone-10, and cytochrome b559 in an approximate 1.3:1:2 molar ratio. Study of the redox poise of these electron carriers indicated that electron flow in the intact complex from unstimulated cells proceeded: NADPH----E-FAD----ubiquinone-10. Similar studies on the complex prepared from stimulated neutrophils indicated that electron flow proceeded: NADPH----E-FAD----ubiquinone-10----cytochrome b559----oxygen. The active enzyme complex was inhibited by p-chloromercuribenzoate. Inhibition persisted after removal of excess inhibitor, was reversed by dithiothreitol, and could be blocked by prior addition of substrate (NADPH). Inhibition of the active oxidase complex by p-chloromercuribenzoate also inhibited electron flow from NADPH to all purported electron carriers in the chain (i.e. E-FAD, ubiquinone-10, and cytochrome b559). We conclude that activation of the oxidase enzyme complex in the intact neutrophil resulted in linkage of electron carrier function between endogenous ubiquinone-10 and cytochrome b559 and was without demonstrable effect on proximal electron flow. The p-chloromercuribenzoate sensitive site(s) proximal to the initial electron acceptor (E-FAD) did not appear to be altered by the cellular activation process.
Asunto(s)
Grupo Citocromo b/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Neutrófilos/enzimología , Complejo de Proteína del Fotosistema II , Ubiquinona/metabolismo , 4-Cloromercuribencenosulfonato/farmacología , Cromatografía Líquida de Alta Presión , Ditiotreitol/farmacología , Activación Enzimática , Humanos , NADPH Oxidasas , Oxidación-ReducciónRESUMEN
The resolved flavoprotein and cytochrome b559 components of the NADPH dependent O2- . generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b559, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent O2- . generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b559, depleted of flavoprotein, demonstrated no measureable NADPH dependent O2- . generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b559 was rapidly oxidized by oxygen, as was the cytochrome b559 in the intact oxidase.
Asunto(s)
Grupo Citocromo b/sangre , Flavoproteínas/sangre , NADH NADPH Oxidorreductasas/sangre , NADPH Oxidasas , Neutrófilos/enzimología , Aerobiosis , Anaerobiosis , Grupo Citocromo b/aislamiento & purificación , Flavoproteínas/aislamiento & purificación , Humanos , Cinética , NADH NADPH Oxidorreductasas/aislamiento & purificación , Espectrometría de FluorescenciaRESUMEN
Control of the intraphagosomal pH in neutrophils may be of importance in creating a microbicidal environment by regulating the activity of the O2-.-generating NADPH oxidase and the lysosomal enzymes discharged into this compartment. In this study, we examined the proton stoichiometry associated with the primary enzymatic reaction underlying the respiratory burst. A preparation of the neutrophil-derived, membrane oxidase consumed NADPH and generated O2-. with a stoichiometry of 1 NADPH:2 O2-. When the enzymatically produced O2-. was prevented from undergoing dismutation, net protons were released in an approximate 1:2 stoichiometry with O2-. generated. In contrast, when O2-. was allowed to dismutate to H2O2, net protons were consumed in a 1:1 stoichiometry with the accumulated H2O2. Thus, the delta pH associated with the NADPH oxidase-dependent production of O2-. was dictated by the fate of the generated radical. The consumption of the oxidase-generated H2O2 by the lysosomal enzyme myeloperoxidase resulted in the formation of HOCl which was trapped in the presence of taurine as the N-chloro derivative. The ratio of chlorinated product formed to H+ consumed was 1:1. The implications of these results are discussed in terms of the known intraphagosomal pH changes that occur following neutrophil stimulation. We conclude that the O2-.-generating oxidase plays a dual role in the phagosome by simultaneously creating an oxidizing environment that optimizes pH-dependent microbicidal processes.
Asunto(s)
Neutrófilos/metabolismo , Consumo de Oxígeno , Fraccionamiento Celular , Peróxido de Hidrógeno/sangre , Concentración de Iones de Hidrógeno , Cinética , NADH NADPH Oxidorreductasas/sangre , NADP/sangre , NADPH Oxidasas , Oxidación-Reducción , Taurina/análogos & derivados , Taurina/sangreRESUMEN
Growth hormone secretagogues (GHSs) represent attractive therapeutic alternatives to recombinant growth hormone (GH), given their ability to amplify pulsatile hormone secretion in a relatively physiologic manner. CP-424,391 (391) is a novel, orally active pyrazolinone-piperidine [corrected] GHS. In rat pituitary cell cultures, 391 stimulated GH release with an EC50 = 3 nM. The addition of 391 to rat pituitary cells activated intracellular calcium signaling but did not elevate intracellular cyclic adenosine monophosphate (cAMP). 391 also modulated the effects of GH-releasing hormone and somatostatin on pituitary cell GH-release and intracellular signaling. In nonpituitary cell lines, the ability of 391 to stimulate intracellular signaling was dependent on the expression of recombinant human GHS receptor. Acute administration of 391 to anesthetized rats or to conscious dogs induced pulsatile release of G H in a dose-dependent manner. Plasma insulin-like growth factor-I (IGF-I) was elevated progressively over a 5-d course of daily oral dosing in dogs. Chronic oral administration of 391 augmented body weight gain in rats and dogs. Thus, the peptidomimetic GHS 391 has potential utility for the treatment of clinical conditions that could benefit from systemic augmentation of GH and IGF-I levels.