RESUMO
A photoaffinity labeling technique is described by which a tetrodotoxin analog is covalently bound to receptor sites associated with the sodium pores of excitable membranes. The biological activity of the toxin analog is retained after the covalent binding reaction.
Assuntos
Marcadores de Afinidade , Músculos/metabolismo , Receptores de Droga/metabolismo , Tetrodotoxina/metabolismo , Marcadores de Afinidade/síntese química , Marcadores de Afinidade/farmacologia , Animais , Bufo marinus , Potenciais da Membrana/efeitos dos fármacos , Fotoquímica , Tetrodotoxina/análogos & derivados , Tetrodotoxina/farmacologiaRESUMO
Lovastatin, a cholesterol-lowering drug, decreased plasma cholesterol and cardiac tissue coenzyme Q10 levels in guinea pigs given 20 mg per kg body weight twice a day. Plasma cholesterol levels were reduced 40% in animals 2 to 4 months of age and 61% in animals 2 years of age. Coenzyme Q10 values in cardiac muscle and cardiac mitochondria of the treated, older group were decreased 31% and 37%, respectively. A significant decrease was not observed in coenzyme Q10 levels of the younger animal group. The potential to phosphorylate ADP to ATP driven by pyruvate-malate and succinate oxidation was decreased 43% and 45%, respectively, for cardiac mitochondria from the treated, 2-year-old animals. A decrease in phosphorylation potential was not observed for the younger group. The respiratory burst of leukocytes isolated from the intraperitoneal cavities of the treated, older animals was decreased 67%, while leukocytes isolated directly from their blood was decreased 76% (Diebold, B., Bhagavan, N. and Guillory, R. (1991) FASEB J. 5, A1203). In contrast to the intact leukocytes, the superoxide production of the cell-free systems prepared from leukocytes isolated from treated and untreated animals did not differ significantly. These observations suggest that in vivo lovastatin may not directly affect the leukocyte superoxide generating system, but may influence it indirectly possibly by modifying the lipid content of the membrane.
Assuntos
Leucócitos/efeitos dos fármacos , Lovastatina/farmacologia , Explosão Respiratória/efeitos dos fármacos , Animais , Colesterol/sangue , Coenzimas , Cobaias , Leucócitos/enzimologia , Masculino , Mitocôndrias Cardíacas/enzimologia , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , Fosforilação , Superóxidos/análise , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
An NADPH dependent cytochrome c reductase has been purified from resting bovine neutrophil membranes. A high degree of purification, approaching homogeneity, is indicated by the presence of a single 75 kDa protein band on silver stained SDS-PAGE (10%). The purified protein catalyzes as well an NADPH dependent reduction of iodonitrotetrazolium violet (INT). Limited papain digestion of the purified preparation produces a 65 kDa product which retains both enzymatic activities. In a similar fashion papain digestion of the plasma membrane bound protein generates a fully active soluble NADPH dependent INT and cytochrome c reductase preparation (65 kDa). Proteolytic cleavage would appear to occur at a protein-membrane anchor remote from the proteins catalytic site. The cytochrome c reductase acts independently of the O2-generating cytochrome b558, a leukocyte plasma membrane protein which also catalyzes an NADPH dependent INT reduction.
Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , NADH Desidrogenase/isolamento & purificação , NADP/metabolismo , Neutrófilos/metabolismo , Papaína/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Grupo dos Citocromos c/metabolismo , Proteínas de Membrana/metabolismo , NADH Desidrogenase/metabolismo , Oxirredução , Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Sais de Tetrazólio/metabolismoRESUMO
Yeast glyceraldehyde-3-phosphate dehydrogenase was labeled in a photodependent reaction by the arylazido-beta-alanyl derivative of NAD+. This analogue was bound covalently to the enzyme and could be reduced in situ by the substrate glyceraldehyde-3-phosphate. That this derivative was bound to the active site in the proper orientation was shown by fluorescence experiments, from the retention of the enzymatic activity when the photolysis of the enzyme-analogue binary complex was carried out in the presence of NAD+. In the dark a non-photodependent competitive inhibition corresponding to a KI-value of 150 microM was observed. Thiol groups of the enzyme were not modified in the photolabeling reaction. Of the various arylazido-beta-alanyl nucleotide derivatives studied, the NADP+ derivative influenced the enzymatic activity to the greatest extent; this is probably due to an ionic bond between enzyme and nucleotide, in addition to the covalent bond of the photolytic reaction.
Assuntos
Marcadores de Afinidade/metabolismo , Azidas , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NAD/análogos & derivados , Sítios de Ligação , NAD/metabolismo , Fotólise , Saccharomyces cerevisiae/enzimologia , Espectrometria de FluorescênciaRESUMO
Following its photolysis in the presence of the isolated guinea-pig vas deferens, the ATP photoaffinity label ANAPP3 produces a specific antagonism of adenine nucleotide-induced contractile responses which are mediated by P2-purinergic receptors. To characterize the site of covalent photoincorporation of ANAPP3, intact vasa deferentia were treated with [3H]ANAPP3 and samples of homogenate, cytosol and a crude membrane fraction were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Photolysis of [3H]ANAPP3 (10(-5) M; 3.0 mu Ci/ml) resulted in the incorporation of radioactivity into cellular components with apparent molecular weights of 54-66 and 43-57 kilodaltons. The photoincorporation of [3H]ANAPP3 was associated with the crude membrane fraction and not the cytosol, was reduced in the presence of ATP in an ATP-concentration-dependent manner, was lessened following pretreatment of the tissues with photolyzed nonradiolabeled ANAPP3, and was unaffected by the nucleoside transport inhibitor, dipyridamole. In tension studies on the same tissues the presence of ATP resulted in a concentration-dependent reduction in the initial contractile response to [3H]ANAPP3 the response to 3H was antagonized in tissues which had been pretreated with nonradiolabeled ANAPP3, and dipyridamole had no effect on the contractile response to [3H]ANAPP3. According to several criteria these findings indicate that the antagonism by photolyzed ANAPP3 of adenine nucleotide-induced responses is a direct result of the covalent insertion at or near the recognition site of cell-surface P2-purinergic receptors.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Marcadores de Afinidade/metabolismo , Azidas/metabolismo , Músculo Liso/metabolismo , Receptores de Neurotransmissores/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Azidas/farmacologia , Dipiridamol/farmacologia , Cobaias , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Fotólise , Receptores de Neurotransmissores/análise , Receptores Purinérgicos , Trítio , Ducto Deferente/metabolismoAssuntos
Tecido Adiposo/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Animais , Antibacterianos/farmacologia , Antimetabólitos/farmacologia , Dinitrofenóis/farmacologia , Ácidos Graxos/metabolismo , Glutaratos/farmacologia , Metabolismo dos Lipídeos , Masculino , Métodos , Norepinefrina/biossíntese , Oligomicinas/farmacologia , Coelhos , Ratos , Soroalbumina Bovina/farmacologia , Succinatos/farmacologia , Terpenos/farmacologiaAssuntos
Antibacterianos/farmacologia , Antimetabólitos/farmacologia , Rhodospirillum/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina , Cromatóforos Bacterianos/metabolismo , Cianetos/farmacologia , Depressão Química , Luz , Fosfatos/metabolismo , Isótopos de Fósforo , Estimulação QuímicaAssuntos
Cromatóforos Bacterianos/metabolismo , Oxirredutases/metabolismo , Rodopseudomonas/enzimologia , Rhodospirillum/enzimologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Cloromercurobenzoatos/farmacologia , Ditiotreitol/farmacologia , Oxirredutases/antagonistas & inibidores , Pirofosfatases/metabolismo , Rodopseudomonas/citologia , Rhodospirillum rubrum/citologia , Rhodospirillum rubrum/enzimologia , VibraçãoAssuntos
Antibacterianos/farmacologia , Antimetabólitos/farmacologia , Mitocôndrias Hepáticas/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Trifosfato de Adenosina , Amobarbital/farmacologia , Animais , Antimicina A/farmacologia , Depressão Química , Manometria , Microscopia Eletrônica , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosfatos , Polarografia , Ratos , Espectrofotometria , Estimulação Química , Estrôncio/farmacologia , Succinatos/metabolismo , TemperaturaAssuntos
Cromatóforos Bacterianos/metabolismo , Rhodospirillum/metabolismo , Trifosfato de Adenosina/farmacologia , Envelhecimento , Sulfato de Amônio , Cromatóforos Bacterianos/efeitos dos fármacos , Cromatóforos Bacterianos/efeitos da radiação , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Precipitação Química , Difosfatos/farmacologia , Ditiotreitol/farmacologia , Transporte de Elétrons , Hidrogenação , Luz , NADP/metabolismo , Efeitos da Radiação , Rhodospirillum rubrum/citologia , Rhodospirillum rubrum/efeitos dos fármacos , Rhodospirillum rubrum/metabolismo , Rhodospirillum rubrum/efeitos da radiação , Frações Subcelulares , Fatores de TempoAssuntos
Trifosfato de Adenosina/análogos & derivados , Marcadores de Afinidade , Azidas , NAD/análogos & derivados , Adenosina Trifosfatases/metabolismo , Marcadores de Afinidade/síntese química , Alanina , Oxirredutases do Álcool/metabolismo , Azidas/síntese química , Sítios de Ligação , Métodos , Miosinas/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fotoquímica , Marcadores de SpinAssuntos
Antibacterianos/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Bovinos , Clorpromazina/farmacologia , Dinitrofenóis/farmacologia , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , RatosAssuntos
Marcadores de Afinidade , NADH NADPH Oxirredutases/metabolismo , Quinona Redutases/metabolismo , Oxirredutases do Álcool/metabolismo , Animais , Azidas , Bovinos , Espectroscopia de Ressonância Magnética , NAD/análogos & derivados , NAD/metabolismo , NAD(P)H Desidrogenase (Quinona) , NADP/análogos & derivados , Fotoquímica , Nucleotídeos de Pirimidina , Ubiquinona/metabolismo , beta-Alanina/análogos & derivadosRESUMO
A cell-free system prepared from polymorphonuclear neutrophils is capable of NADPH-dependent generation of superoxide anion, but requires the simultaneous presence of plasma membranes, cytosol, arachidonate and guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The isolated membranes from such a preparation are able to catalyse NADPH-dependent superoxide formation independently of added cytosol and activators. Such activated membranes, activated in the cell-free system, must consequently contain all of the essential components required by the oxidase for superoxide formation, including the NADPH-binding component. Arylazido-beta-alanyl-[32P]NADPH (3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino] propionyl)-[32P]NADPH), an NADPH analogue and photoaffinity probe, is shown to act in the dark as a substrate for the oxidase activity in the activated membranes and an irreversible photodependent inhibitor following photoirradiation. The photoaffinity probe has been used to identify the specific NADPH-binding component of the oxidase in the activated membranes. In contrast with the sensitivity of the activated membranes, photoirradiation of arylazido-beta-alanyl-[32P]-NADPH under identical conditions, but with non-activated membranes, did not prevent subsequent activation of the treated membranes by cytosol, arachidonate and GTP[S]. However, photoirradiation of the cytosolic fraction in the presence of arylazido-beta-alanyl-[32P]NADPH resulted in an inhibition of the cytosol's ability to activate superoxide generation upon subsequent incubation with plasma membranes in the presence of arachidonate and GTP[S]. These observations are taken as a strong indication that the NADPH-binding protein of the oxidase is a cytosolic factor which associates with the plasma membrane upon activation. The superoxide-generating activity of the activated membranes was inhibited irreversibly in a concentration- and photo-dependent manner by arylazido-beta-alanyl-NADPH. The arylazido-beta-alanyl-NADPH-photodependent inhibition of superoxide generation in the activated membranes correlated with the photodependent labelling of a protein of 55.6 kDa by the arylazido-beta-alanyl-NADPH. Specificity of labelling was indicated by a lack of labelling of the 55.6 kDa region in the non-activated membranes and protection of the photodependent inhibition and labelling of the 55.6 kDa protein by NADPH. It is proposed that the arylazido-beta-alanyl-NADPH-labelled 55.6 kDa protein present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase.
Assuntos
Proteínas de Transporte/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , NADP/metabolismo , Neutrófilos/enzimologia , Superóxidos/metabolismo , Marcadores de Afinidade , Animais , Azidas/metabolismo , Azidas/farmacologia , Sítios de Ligação , Proteínas de Transporte/química , Membrana Celular/enzimologia , Citosol/enzimologia , Cobaias , NADP/análogos & derivados , NADP/farmacologia , Neutrófilos/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Explosão RespiratóriaRESUMO
Polymorphonuclear neutrophil membranes, activated in a cell-free system, contain all of the essential components required for superoxide formation including the NADPH-binding component. Arylazido-beta-alanyl- [32P]NADPH--3'-O-(3-[N-(4-azido-2-nitrophenyl)amino] propionyl)-[32P]NADPH--an NADPH analogue and photoaffinity probe, has been used to identify the specific NADPH binding component of the oxidase in activated membranes. A protein of about 52 kDa was photodependently labeled in the activated membranes by arylazido-beta-alanyl-[32P]NADPH. Specificity of labeling was indicated by the absence of such labeling in nonactivated membranes. The 52-kDa-labeled protein was the only isotopically labeled protein extracted from the labeled membranes with the chaotrope sodium perchlorate. Sodium perchlorate extraction of the 52-kDa protein from activated membranes correlates with the loss of the membranes' superoxide-generating capability. Reconstitution of the lost activity for sodium perchlorate-extracted membranes was accomplished by reincubating the extracted membranes with cytosol. It is proposed that the arylazido- beta-alanyl-[32P]NADPH-labeled protein of 52- to 57-kDa present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase.
Assuntos
NADH NADPH Oxirredutases/química , Neutrófilos/enzimologia , Explosão Respiratória , Marcadores de Afinidade , Animais , Membrana Celular/química , Citosol/química , Flavina-Adenina Dinucleotídeo/metabolismo , Cobaias , Peso Molecular , NADP/metabolismo , NADPH Oxidases , Fotoquímica , Ligação Proteica , Superóxidos/metabolismoRESUMO
Arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) NAD+) is a potent competitive inhibitor with respect to NADH (apparent Ki, 1.7-2.7 microM) for the purified mitochondrial NADH dehydrogenase (EC 1.6.99.3). Upon photoirradiation of the dehydrogenase in the presence of arylazido-beta-[3-3H]-alanyl NAD+, radioactivity was found to be associated with the Mr = 57,000 subunit without significant labeling of what are considered to be the enzyme's two smaller subunits. This labeling could be prevented by the presence of NADH during photoirradiation. In contrast to arylazido-beta-alanyl NAD+, arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]-propionyl) NADP+) did not inhibit dehydrogenase activity nor did photoirradiation of the enzyme in the presence of the radiolabeled analogue (arylazido-beta-[3-3H]alanyl NADP+) result in radioactivity being incorporated into the enzyme. It is concluded that the Mr -- 57,000 subunit contains the pyridine nucleotide-binding site of the mitochondrial NADH electron transport system.