RESUMO
Adhesive interactions between neurons and extracellular matrix (ECM) play a key role in neuronal pattern formation. The prominent role played by the extracellular matrix protein tenascin/cytotactin in the development of the nervous system, tied to its abundance, led us to speculate that brain may contain yet unidentified tenascin receptors. Here we show that the neuronal cell adhesion molecule contactin/F11, a member of the immunoglobulin(Ig)-superfamily, is a cell surface ligand for tenascin in the nervous system. Through affinity chromatography of membrane glycoproteins from chick brain on tenascin-Sepharose, we isolated a major cell surface ligand of 135 kD which we identified as contactin/F11 by NH2-terminal sequencing. The binding specificity between contactin/F11 and tenascin was demonstrated in solid-phase assays. Binding of immunopurified 125I-labeled contactin/F11 to immobilized tenascin is completely inhibited by the addition of soluble tenascin or contactin/F11, but not by fibronectin. When the fractionated isoforms of tenascin were used as substrates, contactin/F11 bound preferentially to the 190-kD isoform. This isoform differs in having no alternatively spliced fibronectin type III domains. Our results imply that the introduction of these additional domains in some way disrupts the contactin/F11 binding site on tenascin. To localize the binding site on contactin/F11, proteolytic fragments were generated and characterized by NH2-terminal sequencing. The smallest contactin/F11 fragment which binds tenascin is 45 kD and also begins with the contactin/F11 NH2-terminal sequence. This implies that contactin/F11 binds to tenascin through a site within the first three Ig-domains.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/química , Embrião de Galinha , Cromatografia de Afinidade , Contactinas , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/química , Imunoglobulinas/química , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Fragmentos de Peptídeos/metabolismo , Ensaio Radioligante , TenascinaRESUMO
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.
Assuntos
Actinina/metabolismo , Biomarcadores , Contração Muscular , Proteínas Musculares/metabolismo , Músculo Liso Vascular/citologia , Citoesqueleto de Actina/ultraestrutura , Actinina/química , Actinina/imunologia , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Diferenciação Celular , Células Cultivadas , Galinhas , Coturnix , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Peso Molecular , Proteínas Musculares/química , Proteínas Musculares/imunologia , Músculo Liso Vascular/química , Peptídeos/químicaRESUMO
Immunohistochemical studies carried out with liver sections show that spectrin is uniformly distributed along the whole plasma membrane of hepatocytes. The bilecanalicular spectrin is released during the purification of liver subplasma membrane fractions, whereas most of the basolateral spectrin remains tightly bound to the membrane. Spectrin associated with the basolateral membranes has been purified and its subunits isolated. The alpha-subunit retains the ability to bind both calmodulin and actin. Fragments have been obtained either by chemical or by proteolytical digestion of the 240 kDa alpha-subunit. Treatment with CNBr yields fragments of about 30 kDa which bind actin and calmodulin. Digestion with Staphylococcus aureus V-8 proteinase yields a calmodulin-binding fragment of 27 kDa and an actin-binding fragment of 31 kDa.
Assuntos
Fígado/análise , Espectrina/isolamento & purificação , Actinas/metabolismo , Animais , Calmodulina/metabolismo , Membrana Celular/análise , Brometo de Cianogênio , Feminino , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Ratos , Serina Endopeptidases , Espectrina/metabolismoRESUMO
Rats of the Milan Hypertensive Strain (MHS) may be considered a useful model for understanding the genetic molecular mechanism underlying a primary form of hypertension in at least a subgroup of patients. Many differences between MHS and its normotensive control strain (MNS) were found at the organ, cellular and biochemical level. In the present investigation renal cell membrane proteins (BBMV) were analysed by two-dimensional electrophoresis and a difference between MHS and MNS was shown in a polypeptide of 32 kDa, subsequently identified as the C-terminal fragment of aminopeptidase M (APM). The activity of the enzyme was higher in MHS. Genetic relationships between this enzyme and the other biochemical cellular abnormalities of MHS, namely sodium transport in BBMV and renin activity in kidney cortex were investigated in MHS, MNS and in two inbred recombinant strains. This analysis showed that faster sodium transport, low kidney levels of renin and hypertension, but not differences in two-dimensional electrophoretic pattern and in aminopeptidase M activity, cosegregated in recombinant strains. These results are consistent with the hypothesis that the faster sodium transport can be considered a primary cellular abnormality responsible for hypertension in MHS and that the aminopeptidase difference is not involved in the cellular abnormalities.
Assuntos
Aminopeptidases/metabolismo , Hipertensão Renal/genética , Córtex Renal/metabolismo , Renina/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico , Pressão Sanguínea , Antígenos CD13 , Eletroforese em Gel Bidimensional , Córtex Renal/ultraestrutura , Cinética , Microvilosidades/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Many cancer patients develop tumor-reactive immune responses against antigens that are either expressed on the surface of tumor cells or released from them into the peripheral circulation. In this study, tumor-reactive immunoglobulins, present in the sera of ovarian cancer patients, were used to identify commonly recognized tumor-associated antigens on ovarian tumor cells. Western immunoblot analysis of cellular proteins, obtained from UL-1 ovarian tumor cell line, demonstrated several commonly recognized immunoreactive proteins. Two of these proteins (Mr 32,000 and 71,000) were selected for further investigation. Cellular proteins isolated from normal human ovarian epithelia, in a similar fashion, failed to exhibit corresponding immunoreactivity to these proteins. As an additional control, sera from normal (nontumor-bearing) individuals failed to identify these proteins on Western immunoblots. Furthermore, the absorption of the ovarian cancer patients' sera with normal ovarian epithelial tissue did not remove the reactivity of these two proteins. The Mr 32,000 and 71,000 proteins were subsequently purified by reverse-phase high-performance liquid chromatography, separated by SDS-PAGE, transferred to the polyvinylidene difluoride membrane, and digested with trypsin. These resulting tryptic fragments were separated by microbore reverse-phase high-performance liquid chromatography, and selected fragments were sequenced by mass spectrometry. This sequence analysis identified the Mr 32,000 protein as cathepsin D and the Mr 71,000 as glucose-regulated protein 78 (member of the heat shock protein family). The identities of cathepsin D and glucose-regulated protein 78 were confirmed by Western blot analysis. Additionally, the presence of cathepsin D was demonstrated in association with immune complexes in vivo. Currently, the common antigenic epitopes of these proteins are being defined.
Assuntos
Adenocarcinoma Mucinoso/imunologia , Adenocarcinoma Papilar/imunologia , Anticorpos Antineoplásicos/biossíntese , Antígenos de Neoplasias/imunologia , Proteínas de Transporte/imunologia , Catepsina D/imunologia , Cistadenoma Papilar/imunologia , Proteínas de Choque Térmico , Chaperonas Moleculares/imunologia , Neoplasias Ovarianas/imunologia , Adenocarcinoma Mucinoso/sangue , Adenocarcinoma Mucinoso/patologia , Adenocarcinoma Papilar/sangue , Adenocarcinoma Papilar/patologia , Idoso , Anticorpos Antineoplásicos/sangue , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/sangue , Western Blotting , Proteínas de Transporte/isolamento & purificação , Catepsina D/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cistadenoma Papilar/sangue , Cistadenoma Papilar/patologia , Chaperona BiP do Retículo Endoplasmático , Epitopos/imunologia , Feminino , Humanos , Espectrometria de Massas , Pessoa de Meia-Idade , Chaperonas Moleculares/isolamento & purificação , Peso Molecular , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Fragmentos de Peptídeos/química , Análise de Sequência , Células Tumorais CultivadasRESUMO
Calpain proteolysis of the plasma membrane Ca2+ pump removes a C-terminal 14-kDa portion which includes the calmodulin-binding domain. This produces a fully activated 124-kDa fragment, which can be inhibited by synthetic versions of the calmodulin-binding domain. The inhibition is strongest when Trp-8 in the latter domain is replaced by a Tyr residue (Falchetto, R., Vorherr, T., Brunner, J., & Carafoli, E., 1991, J. Biol. Chem. 266, 2930-2936). In the present study, the N-terminus of the 28-residue synthetic calmodulin-binding domain was acetylated with 3H-acetic anhydride, and Phe in position 25 was replaced by a phenylalanine derivatized with a diazirine-based, photoactivatable carbene precursor. This peptide (C28WC*) inhibited the fully active 124-kDa fragment of the pump and became cross-linked to it upon photolysis. After proteolysis of the fragment with Asp-N or Staphylococcus aureus V8 (Glu-C) protease, labeled peptides were isolated by reversed-phase high-performance liquid chromatography and subjected to Edman sequence analysis. The peptides originated from a region of the pump located within the unit protruding into the cytoplasm between transmembrane domain two and three. This unit has been proposed to be the site of the energy transduction domain, which would couple the ATP hydrolysis to Ca2+ translocation.
Assuntos
ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/fisiologia , Calmodulina/metabolismo , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/metabolismo , ATPases Transportadoras de Cálcio/isolamento & purificação , Calmodulina/isolamento & purificação , Calpaína/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Endopeptidases , Eritrócitos/enzimologia , Humanos , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
The UDP-N-acetylglucosamine 1-carboxyvinyltransferase (enolpyruvyltransferase, EC 2.5.1.7) which catalyses the first committed step in the biosynthesis of the bacterial cell-wall peptidoglycan was purified to near homogeneity from Enterobacter cloacae and the NH2-terminal amino-acid sequence determined. Using the polymerase chain reaction a 53-bp DNA fragment was synthesized; this fragment encodes the NH2-terminal sequence of the enzyme. A clone was then isolated which contained an open reading frame of 1257 bp coding for a protein of 419 amino acids. This protein was overexpressed 100-fold in transformed Escherichia coli cells and shown to possess the enolpyruvyltransferase activity. The overall amino-acid sequence of the enolpyruvyltransferase is significantly similar to that of the 5-enolpyruvylshikimate 3-phosphate synthase, the only other enzyme known to catalyse the transfer of the enolpyruvate moiety of phosphoenolpyruvate to a substrate.
Assuntos
Alquil e Aril Transferases , Enterobacter cloacae/enzimologia , Transferases/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Dados de Sequência Molecular , Fases de Leitura Aberta , Mapeamento por Restrição , Alinhamento de Sequência , Transferases/metabolismoRESUMO
The extracellular acid protease of Candida tropicalis was purified from the supernatant fraction of culture medium containing bovine serum albumin as nitrogen source and the NH2-terminal amino acid (aa) sequence of the protein was determined. The gene for the acid protease (ACP) was isolated using a pool of synthetic oligonucleotides as a probe and a segment of the deduced aa sequence was found to be in agreement with the NH2-terminal aa sequence of the protein. The deduced aa sequence of ACP is similar to the aa sequence of proteases of the pepsin family. The nucleotide sequence of the 5' portion of this gene revealed a coding sequence for a 60 residue propeptide containing two Lys-Arg amino acid pairs that have been identified as sites for peptidase processing of several exported peptides and proteins. The final Lys-Arg site occurs at the junction with the mature extracellular form of the acid protease.
Assuntos
Ácido Aspártico Endopeptidases/genética , Candida/genética , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/isolamento & purificação , Sequência de Bases , Candida/enzimologia , Clonagem Molecular , DNA Fúngico , Eletroforese em Gel de Poliacrilamida , Genes Fúngicos , Dados de Sequência Molecular , Pepsina A/química , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
The primary structure of the neurohormone crustacean hyperglycemic hormone (CHH-II) was determined by means of enzymatic digestions, manual Edman degradation, and mass spectrometry. CHH-II is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges that connect residues 7-43, 23-39, and 26-52. The peptide has blocked N- and C-termini, and lacks tryptophan, histidine, and methionine. The CHH-I and CHH-II of Procambarus bouvieri have identical sequences and elicit levels of hyperglycemia that are not distinguishable. The difference between the two isomorphs consists in a posttranslational modification of a L-Phe in CHH-I to a D-Phe in CHH-II at the third position from the N-terminus.
Assuntos
Astacoidea/química , Hormônios de Invertebrado/química , Proteínas do Tecido Nervoso/química , Sequência de Aminoácidos , Aminoácidos/química , Animais , Proteínas de Artrópodes , Astacoidea/genética , Astacoidea/metabolismo , Cromatografia Líquida de Alta Pressão , Cisteína/química , Ensaio de Imunoadsorção Enzimática , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , EstereoisomerismoRESUMO
The amino acid sequence of MIH was elucidated by means of digestions with specific proteases, manual Edman degradation, and mass spectrometry. MIH consists of a 72-residue peptide chain (molecular mass 8322 Da) with six cysteines forming three disulfide bridges that connect residues 7-43, 23-39, and 26-52. It has blocked N- and C-termini and lacks tryptophan, histidine, and methionine. MIH shows striking similarity to the crustacean hyperglycemic hormone (CHH) isomorphs of Procambarus bouvieri (90% identity) and to the MIH from Homarus americanus (79% identity) and Penaeus vannamei (46% identity). It is also related to the MIH from Carcinus maenas (28% identity) and Callinectes sapidus (28% identity).
Assuntos
Astacoidea/química , Neuropeptídeos/química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Quimotripsina/metabolismo , Dados de Sequência Molecular , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Análise de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.
Assuntos
Aspergillus fumigatus/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sondas de DNA , Éxons/genética , Biblioteca Gênica , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Serina Endopeptidases/metabolismoRESUMO
Two novel cyclic depsipeptides were isolated from axenic cultures of the terrestrial cyanobacterium Scytonema hofmanni PCC 7110 and designated scyptolin A and B. Amino acid analyses in context with mass and 1H/13C NMR spectroscopies revealed a composition typical for heterologous cyanopeptolins but containing the uncommon residue 3'-chloro-N-methyl-Tyr (cmTyr) and a unique sidechain. Scyptolin A and B both consist of the N-acylated peptide But(1)-Ala(2)-Thr(3)-Thr(4)-Leu(5)-Ahp(6) (3-amino-6-hydroxy-2-oxo-1-piperidine)-Thr(7)-cmTyr(8)-Val(9), which forms a 19-membered ring by esterification of the carboxyl of Val(9) with the hydroxyl of Thr(4). In scyptolin B, the hydroxyl of the Thr(3) residue is additionally esterified with N-butyroyl-Ala. Both scyptolin A and B exhibit selective inhibition of porcine pancreatic elastase in vitro with IC(50) values of 3.1 microg/ml.
Assuntos
Cianobactérias/química , Depsipeptídeos , Inibidores Enzimáticos/isolamento & purificação , Elastase Pancreática/antagonistas & inibidores , Peptídeos Cíclicos/isolamento & purificação , Sequência de Aminoácidos , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Conformação Proteica , SuínosAssuntos
ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/fisiologia , Calmodulina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Calmodulina/metabolismo , Membrana Celular/metabolismo , Modelos Estruturais , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
A synthetic, 28-residue peptide derived from the calmodulin-binding sequence of the plasma membrane Ca2+ pump (C28W) inhibits the ATPase activity of a calpain-produced, truncated fragment of the enzyme. The fragment, which has lost the calmodulin-binding domain, has a molecular mass of 124 kDa and is fully active in the absence of calmodulin. Replacement of Trp-8 in the peptide by an Ala decreases the overall inhibitory activity, while replacement with a Tyr increases it. However, at very low peptide concentrations the effect of Tyr replacement disappears. The synthetic peptide has been made photoactivatable by replacing Phe in position 9 with a synthetic phenylalanine analogue containing a diazirine group and was radioactively labeled by coupling a [3H]acetyl function to its N terminus. After cross-linking with the derivatized peptide, the 124-kDa fragment has been proteolyzed with either Lys-C, Asp-N, or V8 proteases, and the fragment(s) have been separated. Partial sequencing of the cross-linked, radioactive peptides has identified a site of the pump located C terminally to the phosphoenzyme-forming aspartic acid, spanning residues 537-544 of the hPMCA4 isoform of the enzyme. It is concluded that this sequence is part of a site which binds the calmodulin-binding domain of the pump.
Assuntos
Canais de Cálcio/metabolismo , Calmodulina/metabolismo , Sequência de Aminoácidos , Transporte Biológico , ATPases Transportadoras de Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Membrana Eritrocítica/metabolismo , Eritrócitos/enzimologia , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The stimulation of the purified human erythrocyte calcium pump by acidic phospholipids was investigated using synthetic peptides corresponding to a putative phospholipid-responsive domain [Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N. & Carafoli, E. (1990) Biochemistry 29, 8070-8076] and to the calmodulin-binding domain of the pump. The peptides interfered with the activation of the enzyme by phosphatidylserine and phosphatidic acid in competition assays. The peptide corresponding to the calmodulin-binding domain was found to be the most efficient antagonist. Direct binding measurements using fluorescent derivatives of the peptides confirmed the interaction between the acidic phospholipids and the peptides, and fluorescence titrations of dansylated calmodulin with the purified ATPase showed a direct effect of acidic phospholipids on calmodulin binding. A proteolyzed preparation of the Ca(2+)-ATPase lacking the calmodulin-binding domain confirmed that the phospholipid-induced stimulation is mediated by two sites, one located in the C-terminal portion of the previously identified 44-amino-acid phospholipid-responsive domain, the other in the calmodulin-binding domain.
Assuntos
ATPases Transportadoras de Cálcio , Membrana Eritrocítica/metabolismo , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , ATPases Transportadoras de Cálcio/metabolismo , Calmodulina/metabolismo , Cromatografia Líquida de Alta Pressão , Compostos de Dansil/metabolismo , Membrana Eritrocítica/enzimologia , Humanos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Espectrometria de FluorescênciaRESUMO
We have investigated the ability of the neutral ionophore ETH 129 to translocate Ca2+ across artificial and biological membranes. ETH 129 induces Ca2+ transport across planar lipid bilayer. The zero-current membrane potential in a gradient of Ca2+ concentration exhibits Nernst behavior. The dependence of the membrane conductance on ionophore and Ca2+ concentration indicates that three ionophore molecules are needed to transfer one Ca2+ across the hydrophobic region of the membrane. In mitochondria the neutral Ca2+ ionophore can move Ca2+ inside in response to a negative membrane potential under conditions in which the endogenous uniporter is blocked by ruthenium red. This electrophoretic transport of Ca2+ by ETH 129 occurs at a concentration much lower than the one previously reported with the neutral Ca2+ ionophore ETH 1001. Using sea urchin eggs, we have also shown that the efficiency of ETH 129 in inducing egg activation, as revealed by cortical granules exocytosis, is four orders of magnitude higher than that of the commonly used Ca2+ ionophore A21387. ETH 129 is a very efficient and useful tool for use in the investigation of Ca(2+)-dependent biological processes.
Assuntos
Cálcio/metabolismo , Cicloexanos/farmacologia , Ionóforos/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Exocitose/efeitos dos fármacos , Feminino , Técnicas In Vitro , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Transporte de Íons/efeitos dos fármacos , Bicamadas Lipídicas , Membranas Artificiais , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Ratos , Ouriços-do-MarRESUMO
Sphingomonas sp. strain RW1, when grown in salicylate-salts medium, synthesized the enzymes for the degradation of dibenzofuran. The reaction subsequent to meta cleavage of the first benzene ring was found to be catalyzed by two isofunctional hydrolases, H1 and H2, which were purified by chromatography on anion exchange, hydrophobic interaction and gel filtration media. Each enzyme was able to hydrolyze 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate to produce salicylate and benzoate, respectively. SDS/PAGE of each purified enzyme showed a single band of M(r) 31,000 (H1) or 29,000 (H2). The N-terminal amino acid sequences of the two proteins showed 50% homology.
Assuntos
Bacteroidetes/enzimologia , Benzofuranos/metabolismo , Hidrolases/isolamento & purificação , Isoenzimas/isolamento & purificação , Bacteroidetes/genética , Sequência de Bases , Benzoatos/metabolismo , Ácido Benzoico , Biodegradação Ambiental , Catecóis/metabolismo , Ácidos Graxos Insaturados/metabolismo , Hidrolases/genética , Isoenzimas/genética , Dados de Sequência Molecular , Oxigenases/metabolismo , Salicilatos/metabolismo , Ácido SalicílicoRESUMO
A high-throughput screening methodology tailored to the discovery of ligands for known and orphan proteins is presented. With this method, labeling of neither target protein nor screened compounds is required, as the ligands are affinity selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small-molecular-weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in the 96-well format. The protein fraction is analyzed subsequently by liquid chromatography-mass spectrometry for detection and identification of the bound ligand. This screening method was validated with known protein:ligand model systems and optimized for selection of high-affinity binders in an industrial screening environment. All sample handling steps and the analytics are rapid, robust, and largely automated, adopting this technology to the needs of present high-throughput screening processes. This affinity-selection technology, termed SpeedScreen, is currently an integral part of our lead discovery process.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Análise Serial de Proteínas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Ligantes , Espectrometria de Massas/métodos , Ligação Proteica , Proteínas/metabolismoRESUMO
The Ca2+ pumps of the plasma membrane (PM ATPase) and of sarcoplasmic reticulum (SR ATPase) share a number of structural and functional properties. A major difference is the regulatory mechanism. The PM ATPase contains a C-terminal autoinhibitory domain; calmodulin binds to it, removing the inhibition. The SR ATPase contains a domain that interacts with the inhibitor protein phospholamban when the latter is in the nonphosphorylated state; phosphorylation of phospholamban removes the inhibition. Peptides corresponding to the autoinhibitory domain of the PM ATPase were synthesized and found to inhibit the SR ATPase. A 28-residue peptide (C28W), containing the entire autoinhibitory domain, was the most powerful (IC50 = 15 microM; lmax greater than 90%). The inhibition was Ca2+ dependent and more pronounced at submicromolar Ca2+ concentrations. C28W is about 50% homologous to the cytosolic domain of phospholamban, the hydrophilic portion of which was found to interact directly with calmodulin (Kd = about 700 nM). However, while calmodulin reversed the inhibition of the SR ATPase by C28W, it failed to reverse that induced by nonphosphorylated phospholamban.
Assuntos
Proteínas de Ligação ao Cálcio/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/genética , Calmodulina/farmacologia , Membrana Celular/enzimologia , Cães , Cinética , Dados de Sequência Molecular , Músculos/enzimologia , Miocárdio/enzimologia , Peptídeos/síntese química , Fosforilação , Conformação Proteica , Coelhos , Retículo Sarcoplasmático/enzimologiaRESUMO
An increase of a 45 kD protein (p45) in the nuclear matrix has been observed when rat liver cells were proliferatively activated in vivo by a partial hepatectomy. The maximal levels of the association of p45 with the nuclear matrix have been detected 24 hr after hepatectomy just at the time when DNA replication is also maximal. By amino acid sequence analysis, immunoblotting and immunocytochemical methods, it has been demonstrated that p45 is identical to rat cytokeratin D. Immunogold staining of nuclear matrix-intermediate filament preparations from cultured hepatocytes indicated that p45 is associated with cytoskeletal filaments that are strongly interconnected to the lamina, whereas no intranuclear localization of the protein has been detected. With an overlay assay a specific binding of labeled p45 to two nonidentified high-molecular weight proteins and also to lamin B has been observed. Northern blot analysis revealed a biphasic pattern of expression of the messenger RNA for cytokeratin D during liver regeneration. A sharp increase in the messenger RNA levels occurred in the prereplicative phase of liver regeneration a few hours before the accumulation of the protein in the nuclear matrix fraction, and a second peak occurred 48 hr after partial hepatectomy.