RESUMO
Newborn screening (NBS) for Krabbe disease, a rare neurodegenerative disorder caused by deficient galactocerebrosidase (GALC) enzyme activity, has recently been implemented in a number of US states. However, the spectrum of phenotypic manifestations associated with deficient GALC activity complicates the management of screen-positive newborns and underscores the need to identify clinically relevant biomarkers. Earlier studies with a small number of patients identified psychosine, a substrate of the GALC enzyme, as a potential biomarker for Krabbe disease. In this study, we provide, for the first time, longitudinal data on dried blood spot (DBS) psychosine concentrations in different Krabbe disease phenotypes for both untreated patients and those treated with hematopoietic stem cell transplantation (HSCT). Our cohort included patients previously identified by NBS to be at high risk to develop Krabbe disease. Substantially elevated DBS psychosine concentration during the newborn period was found to be a highly specific marker for infantile Krabbe disease. This finding supports the use of DBS psychosine concentration as a second-tier NBS test to aid in the identification of patients who require urgent evaluation for HSCT. In addition, longitudinal assessments showed that both natural disease progression and treatment with HSCT were associated with decreases in DBS psychosine concentrations. Based on these findings we provide recommendations for the interpretation of psychosine concentrations in DBS specimens collected during the first year of life. Future studies should aim to better delineate the relationship between DBS psychosine concentration and disease onset in patients with later-onset forms of Krabbe disease.
Assuntos
Biomarcadores/sangue , Leucodistrofia de Células Globoides/diagnóstico , Psicosina/sangue , Adolescente , Criança , Pré-Escolar , Progressão da Doença , Teste em Amostras de Sangue Seco , Humanos , Lactente , Recém-Nascido , Leucodistrofia de Células Globoides/tratamento farmacológico , Triagem Neonatal , Fenótipo , Espectrometria de Massas em TandemRESUMO
Background Secreted phospholipase A(2) (sPLA(2) ) may be important mediators of asthma, but the specific sPLA(2) s involved in asthma are not known. Objective To evaluate sPLA(2) group IIA, V, and X proteins (sPLA(2) -IIA, sPLA(2) -V, and sPLA(2) -X) in bronchoalveolar lavage (BAL) fluid, BAL cells, and airway epithelial cells of subjects with and without asthma, and examine the relationship between the levels of specific sPLA(2) enzymes and airway inflammation, asthma severity, and lung function. Methods The expression of sPLA(2) -IIA, sPLA(2) -V, and sPLA(2) -X in BAL cells and epithelial brushings was assessed by qPCR. The levels of these sPLA(2) proteins and sPLA(2) activity with and without group II and group X-specific inhibitors were measured in BAL fluid from 18 controls and 39 asthmatics. Results The airway epithelium expressed sPLA(2) -X at higher levels than either sPLA(2) -IIA or sPLA(2) -V, whereas BAL cells expressed sPLA(2) -IIA and sPLA(2) -X at similar levels. The majority of sPLA(2) activity in BAL fluid was attributed to either sPLA(2) -IIA or sPLA(2) -X. After 10-fold concentration of BAL fluid, the levels of sPLA(2) -X normalized to total protein were increased in asthma and were associated with lung function, the concentration of induced sputum neutrophils, and prostaglandin E(2) . The levels of sPLA(2) -IIA were elevated in asthma when normalized to total protein, but were not related to lung function, markers of airway inflammation or eicosanoid formation. Conclusions and Clinical Relevance These data indicate that sPLA(2) -IIA and sPLA(2) -X are the major sPLA(2) s in human airways, and suggest a link between the levels of sPLA(2) -X in the airways and several features of asthma.
Assuntos
Asma/enzimologia , Fosfolipases A2 do Grupo II/metabolismo , Fosfolipases A2 do Grupo X/metabolismo , Sistema Respiratório/enzimologia , Adulto , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Ativação Enzimática/imunologia , Feminino , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Respiratória/metabolismo , Sistema Respiratório/imunologia , Infecções Respiratórias/enzimologia , Infecções Respiratórias/imunologiaRESUMO
Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analyzed by gas-liquid chromatography (GC) and mass spectrometry (MS). This approach revealed that an all-trans geranylgeranyl group was a major isoprenoid modification.
Assuntos
Diterpenos/metabolismo , Proteínas/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Cromatografia Gasosa-Espectrometria de Massas , Células HeLa/metabolismo , Humanos , Ácido Mevalônico/metabolismo , Estrutura Molecular , Níquel , Fragmentos de PeptídeosRESUMO
The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.
Assuntos
Venenos Elapídicos/análise , Fosfatidiletanolaminas/metabolismo , Fosfolipases A/química , Sequência de Aminoácidos , Venenos de Abelha/análise , Sítios de Ligação , Cálcio/metabolismo , Catálise , Fenômenos Químicos , Físico-Química , Cristalização , Ligação de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular , Fosfolipases A/metabolismo , Fosfolipases A2 , Conformação ProteicaRESUMO
The 2.0 angstroms crystal structure of a complex containing bee-venom phospholipase A2 (PLA2) and a phosphonate transition-state analogue was solved by multiple isomorphous replacement. The electron-density map is sufficiently detailed to visualize the proximal sugars of the enzyme's N-linked carbohydrate and a single molecule of the transition-state analogue bound ot its active center. Although bee-venom PLA2 does not belong to the large homologous Class I/II family that encompasses most other well-studied PLA2s, there is segmental sequence similarity and conservation of many functional substructures. Comparison of the bee-venom enzyme with other phospholipase structures provides compelling evidence for a common catalytic mechanism.
Assuntos
Venenos de Abelha/análise , Fosfatidiletanolaminas/metabolismo , Fosfolipases A/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Metabolismo dos Carboidratos , Catálise , Fenômenos Químicos , Físico-Química , Cristalização , Ligação de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular , Fosfolipases A/metabolismo , Fosfolipases A2 , Conformação ProteicaRESUMO
A chemical description of the action of phospholipase A2 (PLA2) can now be inferred with confidence from three high-resolution x-ray crystal structures. The first is the structure of the PLA2 from the venom of the Chinese cobra (Naja naja atra) in a complex with a phosphonate transition-state analogue. This enzyme is typical of a large, well-studied homologous family of PLA2S. The second is a similar complex with the evolutionarily distant bee-venom PLA2. The third structure is the uninhibited PLA2 from Chinese cobra venom. Despite the different molecular architectures of the cobra and bee-venom PLA2s, the transition-state analogue interacts in a nearly identical way with the catalytic machinery of both enzymes. The disposition of the fatty-acid side chains suggests a common access route of the substrate from its position in the lipid aggregate to its productive interaction with the active site. Comparison of the cobra-venom complex with the uninhibited enzyme indicates that optimal binding and catalysis at the lipid-water interface is due to facilitated substrate diffusion from the interfacial binding surface to the catalytic site rather than an allosteric change in the enzyme's structure. However, a second bound calcium ion changes its position upon the binding of the transition-state analogue, suggesting a mechanism for augmenting the critical electrophile.
Assuntos
Fosfolipases A/metabolismo , Venenos de Abelha/análise , Sítios de Ligação , Cálcio/metabolismo , Catálise , Fenômenos Químicos , Físico-Química , Venenos Elapídicos/análise , Modelos Moleculares , Estrutura Molecular , Organofosfonatos/metabolismo , Fosfolipases A/química , Fosfolipases A2 , Fosfolipídeos/metabolismo , Conformação Proteica , Difração de Raios XRESUMO
Phospholipase A2 (PLA2) participates in a wide range of cellular processes including inflammation and transmembrane signaling. A human nonpancreatic secretory PLA2 (hnps-PLA2) has been identified that is found in high concentrations in the synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. This enzyme is secreted from certain cell types in response to the proinflammatory cytokines, tumor necrosis factor or interleukin-1. The crystal structures of the calcium-bound form of this enzyme have been determined at physiological pH both in the presence [2.1 angstrom (A) resolution] and absence (2.2 A resolution) of a transition-state analogue. Although the critical features that suggest the chemistry of catalysis are identical to those inferred from the crystal structures of other extracellular PLA2s, the shape of the hydrophobic channel of hnps-PLA2 is uniquely modulated by substrate binding.
Assuntos
Inflamação/enzimologia , Fosfolipases A/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/química , Gráficos por Computador , Cristalografia , Espaço Extracelular/enzimologia , Humanos , Dados de Sequência Molecular , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Conformação Proteica , Proteínas Recombinantes , Alinhamento de Sequência , Difração de Raios XRESUMO
A method involving electron paramagnetic resonance spectroscopy of a site-selectively spin-labeled peripheral membrane protein in the presence and absence of membranes and of a water-soluble spin relaxant (chromium oxalate) has been developed to determine how bee venom phospholipase A2 sits on the membrane. Theory based on the Poisson-Boltzmann equation shows that the rate of spin relaxation of a protein-bound nitroxide by a membrane-impermeant spin relaxant depends on the distance (up to tens of angstroms) from the spin probe to the membrane. The measurements define the interfacial binding surface of this secreted phospholipase A2.
Assuntos
Glicerofosfolipídeos , Proteínas de Membrana/química , Membranas Artificiais , Fosfolipases A/química , Venenos de Abelha/química , Sítios de Ligação , Cromatos , Espectroscopia de Ressonância de Spin Eletrônica , Lipossomos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Oxalatos , Ácidos Fosfatídicos , Fosfolipases A/análise , Fosfolipases A/genética , Fosfolipases A/metabolismo , Fosfolipases A2 , Marcadores de Spin , Propriedades de SuperfícieRESUMO
Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.
Assuntos
Membrana Celular/metabolismo , Células/metabolismo , Células Eucarióticas/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Proteínas/metabolismo , Animais , Humanos , Laminas , Fator de Acasalamento , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)RESUMO
We have shown previously that interleukin 1 (IL-1) stimulates eicosanoid production in glomerular mesangial cells (MC) by de novo synthesis of a 14-kD, group II phospholipase A2 (PLA2). IL-1-stimulated prostaglandin E2 synthesis precedes expression of this enzyme, suggesting that another PLA2 isoform must be more rapidly activated. In the presence but not absence of calcium inophore, [3H]arachidonate release is increased significantly as early as 5 min after addition of IL-1, and IL-1 concurrently stimulates a Ca(2+)-dependent phospholipase activity, which was characterized as the cytosolic form of PLA2 (cPLA2). IL-1 does not alter either cPLA2 mRNA expression or mass in serum-stimulated MC, suggesting that cPLA2 activity is increased by a posttranslational modification. IL-1 treatment for 30 min doubles 32P incorporation into immunoprecipitable cPLA2 protein, concordant with the increase in enzyme activity. Immunoblot analysis of extracts derived from IL-1-treated (30 min) cells demonstrates a decreased mobility of cPLA2, and treatment of MC lysates with acid phosphatase significantly reduces cytokine-activated cPLA2 activity, further indicating that IL-1 stimulates phosphorylation of the enzyme. IL-1 treatment (24 h) of serum-deprived MC doubled cPLA2 mRNA, protein, and activity. In summary, IL-1 increases cPLA2 activity in a biphasic, time-dependent manner both by posttranslational modification and de novo synthesis. We consider cPLA2 activation a key step in IL-1-stimulated synthesis of pro-inflammatory, lipid mediators, and an integral event in the phenotypic responses induced in target cells by this cytokine.
Assuntos
Mesângio Glomerular/metabolismo , Interleucina-1/farmacologia , Fosfolipases A/metabolismo , Animais , Ácido Araquidônico/metabolismo , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Fosfolipases A/genética , Fosfolipases A2 , Fosforilação , RNA Mensageiro/análise , RatosRESUMO
Secreted phospholipases A(2) have similar catalytic sites, but vastly different interfacial binding surfaces that modulate their binding affinity for different kinds of phospholipid vesicles by several orders of magnitude. The structure/function principles that dictate both the differential interfacial binding and the physiological function of these enzymes are beginning to be unraveled.
Assuntos
Membrana Celular/metabolismo , Fosfolipases A/metabolismo , Animais , Venenos de Abelha/enzimologia , Sítios de Ligação , Cálcio/fisiologia , Catálise , Bovinos , Cristalografia por Raios X , Eicosanoides/metabolismo , Venenos Elapídicos/enzimologia , Humanos , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfolipases A/química , Fosfolipases A/genética , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Suínos , Triptofano/fisiologiaRESUMO
We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.
Assuntos
Marcadores de Afinidade , Marcação por Isótopo , Proteínas/química , Sequência de Aminoácidos , Cromatografia Líquida , Espectrometria de MassasRESUMO
A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A2 (sPLA2s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA2s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA2 was prepared in the structurally native form. The availability of these proteins has allowed kinetic studies to be carried out, to prepare highly selective antisera, to screen for selective inhibitors, to study receptor binding, and to study the action of each enzyme on mammalian cell membranes and their in vivo biological roles.
Assuntos
Expressão Gênica , Fosfolipases A2 Secretórias/metabolismo , Fosfolipídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Clonagem Molecular , Dissulfetos/química , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Fator Xa/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Hidrólise , Corpos de Inclusão/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Fosfolipases A2 Secretórias/química , Fosfolipases A2 Secretórias/genética , Fosfolipídeos/química , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Células Sf9 , Spodoptera , Distribuição TecidualRESUMO
The iron transport protein, transferrin, binds two metal ions and, concomitantly, two carboxylate anions. The metal ion indicators, xylenol orange and semi-xylenol orange are carboxylate anions which exhibit a characteristic visible spectrum when attached to a metal. We prepared the ternary complexes VO2+-transferrin-xylenol orange and VO2+-transferrin-semi-xylenol orange. The EPR spectra show that the vanadyl ion is attached to the protein and the visible spectra show that the xylenol orange or semi-xylenol orange is attached to the metal. The implication is that in other metal-transferrin-anion complexes, the anion is directly attached to the metal.
Assuntos
Glicina/análogos & derivados , Transferrina , Xilenos , Fenômenos Químicos , Química , Espectroscopia de Ressonância Magnética , Fenóis , Sulfóxidos , VanádioRESUMO
For membrane-bound enzymes that act on substrates that partition between the membrane and aqueous phases, it is possible to imagine two fundamentally different mechanisms. Interfacial enzymes must access their substrate from the membrane phase, in other words substrate in the membrane binds directly to the active site of the enzyme at the membrane without mixing with substrate molecules in the aqueous phase. On the other hand, non-interfacial enzymes, either bound to membranes or present in the aqueous phase, must access their substrates from the aqueous phase, i.e. substrate in the aqueous phase binds directly to the enzyme without mixing with substrates in the membrane phase. An interfacial mechanism for some enzymes including secreted and cytosolic phospholipase A(2) and phosphoinositide 3'-hydroxykinase was rigorously proven by demonstrating that these enzymes processively hydrolyze many phospholipids without desorbing from the surface of vesicles (scooting mode). The non-interfacial mechanism is more difficult to establish because it cannot be addressed by steady-state kinetics. Using a pre-steady-state method in which the enzymatic velocity is measured during the time it takes for substrate to exchange between vesicles, a non-interfacial mechanism was proven for vesicle-bound plasma platelet activating factor acetylhydrolase. This enzyme prefers more water-soluble phospholipids such as those with sn-2 acetyl or oxidatively truncated fatty acyl chains, and this is readily explained by the mandatory access of substrate from the aqueous phase.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Membrana Celular/enzimologia , Espaço Extracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipases A/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Sítios de Ligação , Ativação Enzimática , Corantes Fluorescentes , Humanos , Cinética , Fosfatidilcolinas , Fosfolipases A/antagonistas & inibidores , Especificidade por SubstratoRESUMO
A growing number of amphiphiles are known to form high axial ratio microstructures (HARMs) such as the hollow cylindrical microstructures called lipid tubules. As a prelude to exploring the potential of HARMs formed from lipopeptides in controlled release drug delivery, several microstructure formation conditions were investigated. We report the preparation of several glutamic acid dialkyl amides with varying alkyl chain lengths bearing a verity of peptides (1-4 amino acids) [peptide-Glu-(NHCnH2n+1)2, n=12, 14, 16]. These surfactants have been rapidly and efficiently converted into HARMs in aqueous buffer at physiological pH and ionic strength, or in buffer containing MeOH or EtOH. Helical ribbons and tubular HARMs were produced that were stable for as long as 6 months below the phase transition temperatures of the compounds. To estimate the stability of HARMs in vivo, HARMs formed from (Pro)3-Glu(NHC16H33)2 were incubated with DOPC liposomes or fetal calf serum at 40 degreesC. HARM size and shape did not change significantly, suggesting that such lipopeptide particles can retain their morphology long enough in vivo to be useful as drug delivery vehicles.
Assuntos
Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/metabolismo , Peptídeos Cíclicos/metabolismo , Amidas , Varredura Diferencial de Calorimetria , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Estabilidade de Medicamentos , Luz , Lipoproteínas/síntese química , Lipoproteínas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Peptídeos Cíclicos/síntese química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Espalhamento de RadiaçãoRESUMO
We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.
Assuntos
Ácido Araquidônico/metabolismo , Inibidores Enzimáticos/farmacologia , Fosfolipases A/antagonistas & inibidores , Pirrolidinas/farmacologia , Animais , Ácido Araquidônico/análise , Células CHO , Linhagem Celular , Cricetinae , Citosol/enzimologia , Fosfolipases A2 do Grupo IV , Humanos , Macrófagos Peritoneais/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Camundongos , Estrutura Molecular , Fosfolipídeos/metabolismo , Pirrolidinas/químicaRESUMO
Bee venom phospholipase A2 (BV-PLA2) is a hydrolytic enzyme that specifically cleaves the sn-2 acyl bond of phospholipids at the lipid/water interface. The same enzyme is also believed to be responsible for some systemic anaphylactic reactions in bee venom sensitized individuals. To study the structure/function relationships of this enzyme and to define the molecular determinants responsible for its allergenic potential, a synthetic gene encoding the mature form of BV-PLA2 was expressed in Escherichia coli. This enzyme was produced as a fusion protein with a 6xHis-tag on its amino-terminus yielding 40-50 mg of fusion protein per 1 of culture after metal ion affinity chromatography. A kallikrein protease recognition site was engineered between the 6xHis-tag and the amino-terminus of the enzyme allowing isolation of the protein with its correct N-terminus. Recombinant affinity purified BV-PLA2 was refolded, purified to homogeneity, and cleaved with kallikrein, resulting in a final yield of 8-9 mg of active enzyme per 1 of culture. The enzymatic and immunological properties of the recombinant BV-PLA2 are identical to enzyme isolated from bee venom indicating a native-like folding of the protein.
Assuntos
Venenos de Abelha/genética , Fosfolipases A/genética , Sequência de Aminoácidos , Sequência de Bases , Venenos de Abelha/enzimologia , Cromatografia por Troca Iônica , Clonagem Molecular , DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Genes Sintéticos , Calicreínas/metabolismo , Cinética , Dados de Sequência Molecular , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismo , Fosfolipases A2 , Plasmídeos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.
Assuntos
Inibidores Enzimáticos/farmacologia , Cetonas/farmacologia , Organofosfonatos/farmacologia , Fosfolipases A/antagonistas & inibidores , Inibidores Enzimáticos/química , Humanos , Técnicas In Vitro , Cetonas/química , Cinética , Lipossomos , Membranas Artificiais , Micelas , Organofosfonatos/química , Fosfolipases A/classificação , Relação Estrutura-Atividade , Células U937RESUMO
A specific set of proteins in eukaryotic cells contain covalently attached carboxy-terminal prenyl groups (15-carbon farnesyl and 20-carbon geranylgeranyl). Many of them are signaling proteins including Ras, heterotrimeric G proteins and Rab proteins. The protein prenyltransferases which attach prenyl groups to proteins have been well characterized, and an X-ray structure is available for protein farnesyltransferase. Inhibitors of protein farnesyltransferase are showing sufficient promise in preclinical trials as anti-cancer drugs to warrant widespread interest in the pharmaceutical industry.