RESUMO
P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer's disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD analog, [11C]9) and 5-(3-Bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) were prepared from their corresponding desmethylated precursors with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 30-50% decay corrected radiochemical yields with 370-1110GBq/µmol specific activity at EOB. 5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]F-5-BDBD, [18F]5a) and 5-(3-(2-[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]FE-5-BDBD, [18F]11) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[18F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5-25% decay corrected radiochemical yields with 111-740GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD.
Assuntos
Azirinas/química , Di-Hidropiridinas/química , Compostos Radiofarmacêuticos/síntese química , Receptores Purinérgicos P2X4/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Azirinas/síntese química , Azirinas/metabolismo , Ligação Competitiva , Radioisótopos de Carbono/química , Di-Hidropiridinas/síntese química , Di-Hidropiridinas/metabolismo , Radioisótopos de Flúor/química , Células HEK293 , Humanos , Marcação por Isótopo , Tomografia por Emissão de Pósitrons , Ligação Proteica , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Receptores Purinérgicos P2X4/química , Receptores Purinérgicos P2X4/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.
Assuntos
Azirinas/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/enzimologia , Sondas Moleculares/metabolismo , Fosfatidilcolinas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Membrana Celular/efeitos dos fármacos , Quimotripsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Ácido Oleico/farmacologia , Ácidos Fosfatídicos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína , Deleção de Sequência/efeitos dos fármacos , Titulometria , Tosilina Clorometil Cetona/farmacologiaRESUMO
The accumulation of Ca2+ in the mitochondrial matrix can stimulate oxidative phosphorylation, but can also, at high Ca2+ concentrations, transmit and amplify an apoptotic signal. Here, we characterized the capacity of physiological stimuli (for example, histamine and inositol-1,4,5-triphosphate) and inducers of endoplasmic reticulum (ER) stress (for example, A23187, thapsigargin and tunicamycin) to release Ca2+ from ER stores, induce mitochondrial Ca2+ accumulation, and trigger cell death in human cervix and colon carcinoma cell lines. Sustained Ca2+ accumulation in the mitochondrial matrix induced by ER stress triggered signs of proapoptotic mitochondrial alteration, namely permeability transition, dissipation of the electrochemical potential, matrix swelling, relocalization of Bax to mitochondria and the release of cytochrome c and apoptosis-inducing factor from mitochondria. In contrast, rapid and transient accumulation of Ca2+ induced by physiological stimuli failed to promote mitochondrial permeability transition and to affect cell viability. The specificity of this apoptosis pathway was validated in cells using a panel of pharmacological agents that chelate Ca2+ (BAPTA-AM) or inhibit inositol-1,4,5-trisphosphate receptor (IP(3)R; 2-aminoethoxydiphenyl borate), voltage-dependent anion channel (VDAC) (4,4'-diisothiocyanatostilbene-2,2'-disulfonate, NADH), the permeability transition pore (cyclosporin A and bongkrekic acid), caspases (z-VAD-fmk) and protein synthesis (cycloheximide). Finally, we designed an original cell-free system in which we confronted purified mitochondria and ER vesicles, and identified IP(3)R, VDAC and the permeability transition pore as key proteins in the ER-triggered proapoptotic mitochondrial membrane permeabilization process.
Assuntos
Apoptose , Sinalização do Cálcio , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Mitocondriais/metabolismo , Azirinas/metabolismo , Linhagem Celular Tumoral , Sistema Livre de Células , Retículo Endoplasmático/efeitos dos fármacos , Histamina/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Dilatação Mitocondrial , Permeabilidade/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of a model transmembrane peptide. When this peptide was incorporated into lipid bilayers and irradiated with UV light, a covalent bond was formed between the crosslinking probe and a lipid. This crosslinking reaction could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resulting product could be characterized by mass spectrometry. By use of phospholipases, it was demonstrated that the peptide crosslinks to both acyl chains of the lipids. The peptide showed a clear preference to partition into fluid lipids and was excluded from lipids in the gel phase. However, when the peptide was incorporated into bilayers containing two lipid species with different acyl chain lengths, molecular sorting of the lipids around the peptide based on hydrophobic matching was not observed. It is proposed that the size of the transmembrane part plays an important role in the dynamic interactions of membrane proteins with the surrounding lipids and hence in determining whether molecular sorting can occur.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Raios Ultravioleta , 1,2-Dipalmitoilfosfatidilcolina/química , Sequência de Aminoácidos , Azirinas/metabolismo , Dimiristoilfosfatidilcolina/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Proteínas de Membrana/síntese química , Dados de Sequência Molecular , Peptídeos/síntese química , Fenilalanina/análogos & derivados , Marcadores de Fotoafinidade/metabolismo , Fotoquímica , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
To identify regions of the Torpedo Na,K-ATPase alpha-subunit that interact with membrane lipid and to characterize conformationally dependent structural changes in the transmembrane domain, we have proteolytically mapped the sites of photoincorporation of the hydrophobic compounds 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) and the phosphatidylcholine analogue [(125)I]TIDPC/16. The principal sites of [(125)I]TIDPC/16 labeling were identified by amino-terminal sequence analysis of proteolytic fragments of the Na,K-ATPase alpha-subunit and are localized to hydrophobic segments M1, M3, M9, and M10. These membrane-spanning segments have the greatest levels of exposure to the lipid bilayer and constitute the bulk of the lipid-protein interface of the Na,K-ATPase alpha-subunit. The extent of [(125)I]TID and [(125)I]TIDPC/16 photoincorporation into these transmembrane segments was the same in the E(1) and E(2) conformations, indicating that lipid-exposed segments located at the periphery of the transmembrane complex do not undergo large-scale movements during the cation transport cycle. In contrast, for [(125)I]TID but not for [(125)I]TIDPC/16, there was enhanced photoincorporation in the E(2) conformation, and this component of labeling mapped to transmembrane segments M5 and M6. Conformationally sensitive [(125)I]TID photoincorporation into the M5 and M6 segments does not reflect a change in the levels of exposure of these segments to the lipid bilayer as evidenced by the lack of [(125)I]TIDPC/16 labeling of these two segments in either conformation. These results suggest that [(125)I]TID promises to be a useful tool for structural characterization of the cation translocation pathway and for conformationally dependent changes in the pathway. A model of the spatial organization of the transmembrane segments of the Na,K-ATPase alpha- and beta-subunits is presented.
Assuntos
Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Marcadores de Fotoafinidade/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Torpedo , Animais , Azirinas/metabolismo , Endopeptidases/metabolismo , Hidrólise , Radioisótopos do Iodo , Lipídeos de Membrana/química , Proteínas de Membrana/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Fosfatidilcolinas/metabolismo , Fotoquímica , Conformação Proteica , ATPase Trocadora de Sódio-Potássio/química , Relação Estrutura-AtividadeRESUMO
Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEG membrane protein complex. SecA translocation ATPase activity as well as protein translocation is dependent on the presence of negatively charged lipids. By using a phospholipid with an acyl chain linked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored. SecA bound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labeling by the photoactivatable phospholipid. The presence of an excess amount of SecYEG complex resulted in a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-bound form of SecA. These data demonstrate that the SecYEG-bound SecA is largely shielded from the phospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA that differ in the degree of lipid association.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Azirinas/metabolismo , Proteínas de Bactérias/química , Transporte Biológico , Fracionamento Químico , Radioisótopos do Iodo , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/química , Proteolipídeos/metabolismo , Proteolipídeos/fisiologia , Canais de Translocação SEC , Proteínas SecARESUMO
alpha-SNAP [soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein] is required for fusion of transport vesicles with their target membrane. In this study, we have examined the membrane-binding properties of alpha-SNAP. We have found that in several tissues a much larger amount of alpha-SNAP per unit weight of protein is bound to membranes than is free in the cytosol. Biochemical analysis shows that a fraction of alpha-SNAP behaves in ways characteristic of hydrophobic, lipid-associated proteins. These findings suggest that membrane binding may be accounted for, at least in part, by interaction with membrane lipid. Consistent with this idea, binding of newly synthesized alpha-SNAP to brain membranes was found to be independent of functional SNAP receptors and could be accounted for by direct binding of alpha-SNAP to membrane lipid. Furthermore, membrane lipid enhanced the ability of alpha-SNAP to stimulate NSF-dependent ATPase activity.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Adenosina Trifosfatases/metabolismo , Animais , Azirinas/metabolismo , Western Blotting , Encéfalo/metabolismo , Proteínas de Transporte/química , Reagentes de Ligações Cruzadas/metabolismo , Eletroforese em Gel de Poliacrilamida , Cinética , Lipossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/química , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Suínos , Tripsina/metabolismoRESUMO
SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion. Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane. To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m[125I]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles. The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain. We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Proteínas de Escherichia coli , Lipídeos de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Adenosina Trifosfatases/química , Adenilil Imidodifosfato/metabolismo , Azirinas/metabolismo , Proteínas de Bactérias/química , Benzoatos/metabolismo , Ácido Benzoico , Sítios de Ligação , Transporte Biológico , Reagentes de Ligações Cruzadas/metabolismo , Escherichia coli , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Canais de Translocação SEC , Proteínas SecARESUMO
Interleukin 1 is the prototype of an inflammatory cytokine, and evidence suggests that it uses the sphingomyelin pathway and ceramide production to trigger mitogen-activated protein kinase (MAPK) activation and subsequent gene expression required for acute inflammatory processes. To identify downstream signaling targets of ceramide, a radioiodinated photoaffinity labeling analog of ceramide ([125I] 3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramide) was employed. It is observed that ceramide specifically binds to and activates protein kinase c-Raf, leading to a subsequent activation of the MAPK cascade. Ceramide does not bind to any other member of the MAPK module nor does it bind to protein kinase C-zeta. These data identify protein kinase c-Raf as a specific molecular target for interleukin 1 beta-stimulated ceramide formation and demonstrate that ceramide is a lipid cofactor participating in regulation of c-Raf activity.
Assuntos
Azirinas/metabolismo , Ceramidas/metabolismo , Ceramidas/farmacologia , Mesângio Glomerular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Marcadores de Afinidade , Animais , Azirinas/isolamento & purificação , Sítios de Ligação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Ceramidas/isolamento & purificação , Ativação Enzimática , Interleucina-1/farmacologia , Cinética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-raf , RatosRESUMO
The ligand binding domain of the human estrogen receptor (hER-LBD), encompassing the sequence MDPS282AG...V595, has been expressed at high levels in Escherichia coli from a pET-23d vector, and a purified preparation has been characterized both by mass spectrometry and biochemical methods. Inclusion bodies from the bacterial expression were solubilized by sonication and the hER-LBD was purified to near homogeneity by affinity chromatography over an estradiol-Sepharose column in urea-containing buffer. This material ran as a single peak on reversed-phase HPLC, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band with apparent molecular mass of 31-32 kilodaltons (kDa), somewhat smaller than that expected from the construct (35.6 kDa). Edman degradation revealed a single sequence of MDPSAGDMRA, consistent with an intact N terminus. Further characterization of this material using low resolution matrix-assisted laser desorption ionization mass spectrometry indicated an apparent single protein species of average mass 33,143 daltons (Da). Detailed molecular analysis by electrospray ionization mass spectrometry, however, revealed that this purified hER-LBD preparation was actually composed of a number of both modified and unmodified LBD protein fragments between 32,900-33,400 Da. The bulk of this 33-kDa protein mixture consisted of three LBD protein fragments with C termini at Ala571 (70%), Ala569 (23%), and Ser575 (4%), with only a trace amount (3%) of the full length expressed LBD (MDPS282...V595; 35, 602 Da). These four protein species also appear to be partially chemically modified by carbamylation. The functional integrity of this hER-LBD preparation was investigated by a ligand-exchange assay, which showed that the hER-LBD retained full estradiol-binding capacity; specific, covalent labeling was also observed using either the electrophilic affinity-labeling ligand tamoxifen aziridine (TAZ) or the photoaffinity-labeling ligand hexestrol diazirine. Thus, this expressed hER-LBD preparation, while appearing nominally pure by conventional biochemical techniques and having the expected ligand-binding capacity, was shown by sensitive high resolution electrospray ionization mass spectrometry techniques to be largely truncated 20-26 amino acids from the expected C terminus and to have a substantial level of covalent modification arising from the urea.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Espectrometria de Massas , Receptores de Estrogênio/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Azirinas/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Escherichia coli , Estradiol/metabolismo , Hexestrol/análogos & derivados , Hexestrol/metabolismo , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Ligação Proteica , Receptores de Estrogênio/genética , Receptores de Estrogênio/isolamento & purificação , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismoRESUMO
Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.
Assuntos
Tecido Adiposo/metabolismo , Azirinas/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Ácidos Oleicos/metabolismo , Células 3T3 , Animais , Transporte Biológico , Proteínas de Transporte/isolamento & purificação , Diferenciação Celular , Membrana Celular/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Cinética , Camundongos , Peso Molecular , Ácido OleicoRESUMO
To identify regions that are involved in the formation of the dihydropyridine receptor site of skeletal muscle L-type Ca2+ channels, the alpha 1 subunit of the channel complex was specifically labeled with the 1,4-dihydropyridine-receptor-selective photoaffinity probe [3H]diazipine. Photoaffinity-labeled regions were identified by probing labeled proteolytic fragments with several anti-peptide antibodies recognizing different segments of the alpha 1 sequence. Forty to 50% of the alpha 1-associated [3H]diazipine label was contained in the tryptic fragment between Arg-988 and Ala-1023 derived from the loop between segments S5 and S6 in domain III. This region corresponds to a portion of the channel that is believed to contribute to formation of the transmembrane pore. Twenty to 30% of the labeling occurred in a V8 protease fragment between Glu-1349 and Trp-1391. This fragment contains transmembrane segment S6 of domain IV and has previously been shown to form part of the drug receptor for phenylalkylamine Ca2+ antagonists. Our data suggest that the dihydropyridine receptor is formed by close apposition of two discontinuous regions of the alpha 1 subunit sequence in domains III and IV. In light of previous work localizing this receptor site to the extracellular surface of the lipid bilayer, it is proposed that amino acid residues at the extracellular surface in the loop connecting segments IIIS5 and IIIS6 and at the extracellular end of segment IVS6 contribute to formation of the dihydropyridine receptor site.
Assuntos
Azirinas/metabolismo , Canais de Cálcio/metabolismo , Di-Hidropiridinas/metabolismo , Músculos/fisiologia , Receptores Nicotínicos/metabolismo , Marcadores de Afinidade/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Membrana Celular/metabolismo , Bicamadas Lipídicas , Substâncias Macromoleculares , Modelos Estruturais , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Conformação Proteica , CoelhosRESUMO
A 70-kDa lens membrane polypeptide (MP70) is a specific component of the fiber gap junctions. The C-terminal portion of MP70 is removed by age-related proteolytic processing, leaving an N-terminal 38-kDa polypeptide (MP38) in the membrane. Membrane association and topology of MP70 and of its processed form MP38 have been studied by hydrophobic labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and phenyl isothio[14C]cyanate. Membrane-embedded segments have been identified. They are localized in the N-terminal 30-kDa portion of MP70 and MP38. The C-terminal 40-kDa portion of MP70 appears to be exposed entirely at the cytoplasmic side of the junctional membranes. Hence, potentially poreforming peptide segments in the MP70 molecule are conserved upon age-related processing to MP38.
Assuntos
Cristalino/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Azirinas/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Conexinas , Isotiocianatos , Cinética , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência do Ácido Nucleico , Ovinos , Tiocianatos/metabolismoRESUMO
2,5-Bis(1-aziridinyl)-1,4-benzoquinone (BABQ) is a compound from which several antitumour drugs are derived, such as Trenimone, Carboquone and Diaziquone (AZQ). The mechanism of DNA binding of BABQ was studied using 14C-labeled BABQ and is in agreement with reduction of the quinone moiety and protonation of the aziridine ring, followed by ring opening and alkylation. The one-electron reduced (semiquinone) form of BABQ alkylates DNA more efficiently than two-electron reduced or non reduced BABQ. Covalent binding to polynucleotides did not unambiguously reveal preference for binding to specific DNA bases. Attempts to elucidate further the molecular structure of DNA adducts by isolation of modified nucleosides from enzymatic digests of reacted DNA failed because of instability of the DNA adducts. The mechanism of covalent binding to protein (bovine serum albumin, BSA) appeared to be completely different from that of covalent binding to DNA. Binding of BABQ to BSA was not enhanced by reduction of the compound and was pH dependent in a way that is opposite to that of DNA alkylation. Glutathione inhibits binding of BABQ to BSA and forms adducts with BABQ in a similar pH dependence as the protein binding. The aziridine group therefore does not seem to be involved in the alkylation of BSA. Incubation of intact E. coli cells, which endogenously reduce BABQ, resulted in binding to both DNA and RNA, but also appreciable protein binding was observed.
Assuntos
Antineoplásicos/metabolismo , Aziridinas , Azirinas/metabolismo , Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , DNA Bacteriano/metabolismo , DNA/metabolismo , Alquilação , Animais , Bovinos , Cisteína/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Glutationa/farmacologia , Concentração de Íons de Hidrogênio , Oxirredução , RNA Bacteriano/metabolismo , Soroalbumina Bovina/metabolismo , Fatores de TempoRESUMO
A carbene-generating 14C-labelled beta-methoxyacrylate derivative ((E)-methyl-3-methoxy-2-[4-(3-trifluoromethyl-3-diazirinyl) benzoyloxyphenyl] propanoate, uniformly labelled with 14C in the benzene ring of the benzoyl group) has been used to locate the proteins involved in binding this class of inhibitors to bovine heart mitochondrial ubihydroquinone:cytochrome c oxidoreductase. The beta-methoxyacrylate photoaffinity label was shown to be a competent inhibitor of electron transport through the protein complex. Under illumination through a narrow bandpass filter, allowing specific photolysis of the diazirine group, the compound bound to cytochrome b and weakly to an 8 kDa polypeptide. Apart from some binding to a cytochrome b aggregate, other proteins were left unlabelled. The binding could be prevented in the presence of excess amounts of unlabelled beta-methoxyacrylate, myxothiazol or stigmatellin but not by antimycin A or HQNO. At high concentrations DBMIB partially competed for the binding site. The binding site for this class of inhibitors is therefore the 'o-site'. Our results indicate that this site is comprised of residues from cytochrome b and possibly the 8 kDa polypeptide and that the site may be close to the Reiske iron-sulphur protein.
Assuntos
Acrilatos/metabolismo , Azirinas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/metabolismo , Marcadores de Afinidade , Animais , Sítios de Ligação , Bovinos , Fenômenos Químicos , Química , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Luz , Mitocôndrias Cardíacas/enzimologia , Fotoquímica , Análise EspectralRESUMO
The hypoxic cell cytotoxins SR 4233, benznidazole (Benzo), and CB 1954 were readily reduced by anaerobic mouse liver microsomes in vitro to their respective amino or single N-oxide derivatives. The reactions were inhibited in air and required reduced cofactors, particularly NADPH. The rates of reductive bioactivation were markedly different for each drug, with SR 4233 much greater than CB 1954 greater than Benzo. Using purified cytochrome P-450 reductase (P-450 reductase) and an inhibitory antibody to this enzyme, we demonstrated that P-450 reductase was involved in the reductive bioactivation of all 3 compounds. It had a minor role in SR 4233 reduction, but a more important involvement in CB 1954 metabolism to its 4-amino metabolite. Using carbon monoxide, a specific inhibitor of cytochrome P-450 (P-450), we demonstrated that P-450 was involved in both SR 4233 and Benzo reduction. P-450 had a major role both in SR 4233 conversion to SR 4317 and in the latter steps of Benzo amine formation. Purified xanthine oxidase was shown to reduce SR 4233 and Benzo in vitro, but cytosolic aldehyde oxidase activity was only detectable with Benzo as substrate. Characterizing the relative participation of the various reductases in tumor versus normal tissues may allow a more rational selection and application of hypoxic cell cytotoxins in cancer therapy.
Assuntos
Antineoplásicos/metabolismo , Aziridinas/metabolismo , Azirinas/metabolismo , Microssomos Hepáticos/enzimologia , Nitroimidazóis/metabolismo , Radiossensibilizantes/metabolismo , Triazinas/metabolismo , Animais , Biotransformação , Monóxido de Carbono/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/farmacologia , Citosol/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Oxirredução , TirapazaminaRESUMO
Bioreduction in the activation of diaziquone (2,5-diaziridinyl-3,6-bis (carboethoxyamino)-1,4-benzoquinone) has been investigated by exploring its reduction by whole cells, rat liver microsomes and purified enzymes. The mechanism of bioreduction was further investigated by exploring the chemical and electrochemical reduction of diaziquone as well as its photochemistry. Reduced diaziquone (by several means) was then tested for activity against parent compound. It appears that reduced diaziquone in most cases is more active than the oxidized form. Diaziquone redox cycles, but it is easily reduced to the hydroquinone which oxidizes to the semiquinone yielding free radicals under aerobiosis. The most probable mechanism of action is that of bioreductive alkylation where the alkylating aziridines are protonated after reduction facilitating the opening of the aziridine rings and thus alkylation.
Assuntos
Antineoplásicos/metabolismo , Aziridinas/metabolismo , Azirinas/metabolismo , Benzoquinonas , Biotransformação , Radicais Livres , Microssomos Hepáticos/metabolismo , Animais , Humanos , OxirreduçãoRESUMO
A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.
Assuntos
Antineoplásicos/metabolismo , Aziridinas/metabolismo , Azirinas/metabolismo , Carcinoma 256 de Walker/enzimologia , Nitrorredutases/metabolismo , Oxirredutases/metabolismo , Quinona Redutases/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Aziridinas/farmacologia , Biotransformação , Carcinoma 256 de Walker/metabolismo , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona) , Nitrorredutases/antagonistas & inibidores , Quinona Redutases/antagonistas & inibidores , Espectrofotometria UltravioletaRESUMO
Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.
Assuntos
Antineoplásicos/metabolismo , Aziridinas/metabolismo , Azirinas/metabolismo , Carcinoma 256 de Walker/metabolismo , Reagentes de Ligações Cruzadas , Dano ao DNA , Nitrorredutases/metabolismo , Oxirredutases/metabolismo , Animais , Aziridinas/toxicidade , Carcinoma 256 de Walker/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , NAD/metabolismo , Ratos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, alpha-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.