RESUMEN

Two-dimensional crystals with angstrom-scale pores are widely considered as candidates for a next generation of molecular separation technologies aiming to provide extreme, exponentially large selectivity combined with high flow rates. No such pores have been demonstrated experimentally. Here we study gas transport through individual graphene pores created by low intensity exposure to low kV electrons. Helium and hydrogen permeate easily through these pores whereas larger species such as xenon and methane are practically blocked. Permeating gases experience activation barriers that increase quadratically with molecules' kinetic diameter, and the effective diameter of the created pores is estimated as ∼2 angstroms, about one missing carbon ring. Our work reveals stringent conditions for achieving the long sought-after exponential selectivity using porous two-dimensional membranes and suggests limits on their possible performance.

RESUMEN

Despite being only one-atom thick, defect-free graphene is considered to be completely impermeable to all gases and liquids1-10. This conclusion is based on theory3-8 and supported by experiments1,9,10 that could not detect gas permeation through micrometre-size membranes within a detection limit of 105 to 106 atoms per second. Here, using small monocrystalline containers tightly sealed with graphene, we show that defect-free graphene is impermeable with an accuracy of eight to nine orders of magnitude higher than in the previous experiments. We are capable of discerning (but did not observe) permeation of just a few helium atoms per hour, and this detection limit is also valid for all other gases tested (neon, nitrogen, oxygen, argon, krypton and xenon), except for hydrogen. Hydrogen shows noticeable permeation, even though its molecule is larger than helium and should experience a higher energy barrier. This puzzling observation is attributed to a two-stage process that involves dissociation of molecular hydrogen at catalytically active graphene ripples, followed by adsorbed atoms flipping to the other side of the graphene sheet with a relatively low activation energy of about 1.0 electronvolt, a value close to that previously reported for proton transport11,12. Our work provides a key reference for the impermeability of two-dimensional materials and is important from a fundamental perspective and for their potential applications.

RESUMEN

OBJECTIVES: To analyze the related pathogenicity gene mutations in a sudden death of hypertrophic cardiomyopathy （HCM） on whole exome level. METHODS: Whole exome sequencing （WES） was been performed on a sudden death case sample with pathological features of HCM by Illumina® Hiseq 2500 platform. Using hg19 as the reference sequences, the sequencing data were analyzed. Suspicious single nucleotide variants （SNV） were screened, and the conservatism and function were analyzed by the software such as PhyloP, PolyPhen-2, SIFT, etc. RESULTS: After screening, a heterozygous mutation C719R was finally identified in the gene MYBPC3 of this case. CONCLUSIONS: The molecular anatomy on whole exome level by second generation sequencing technology can help to define the molecular mechanism of HCM and provide a new mothed and thought for analysis of death cause.

Asunto(s)

Cardiomiopatía Hipertrófica/mortalidad , Muerte Súbita , Secuenciación del Exoma , Exoma/genética , Pruebas Genéticas/métodos , Adulto , Cardiomiopatía Hipertrófica/genética , Femenino , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Mutación

RESUMEN

We examined polymorphism of the TCTA tetranucleotide sequence in the 3rd intron of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the Han population of Ningxia Province in China. We also looked for a possible relationship between STR polymorphism in the 3rd intron of the HPRT gene and primary hyperuricemia. We used Chelex-100 to extract DNA, then PCR, PAGE and silver staining for allele genotyping and DNA sequencing to obtain the distribution of the alleles. We found, for the first time, that there is high STR polymorphism in the 3rd intron of the HPRT gene. We detected 5 STR alleles in this intron in the Han population of Ningxia Province, with 15 genotypes in females; significant differences were observed in the distribution of alleles and genotypes between control and patient groups for both males and females. Alleles of the TCTA repeat in the 3rd intron of the HPRT gene were found to be associated with primary hyperuricemia; consequently, these alleles may be considered risk factors for primary hyperuricemia.

Asunto(s)

Pueblo Asiatico , Genética de Población , Hiperuricemia/genética , Hipoxantina Fosforribosiltransferasa/genética , Repeticiones de Microsatélite , Polimorfismo Genético , Alelos , Secuencia de Bases , China , Femenino , Frecuencia de los Genes , Genotipo , Técnicas de Genotipaje , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Factores de Riesgo

RESUMEN

Reelin is an extracellular signaling protein that plays an important role in the development of the central nervous system. Post-mortem studies have shown lower reelin protein levels in the brains of patients with schizophrenia and bipolar disorder compared with controls. Genetic studies have also shown that mutations in the reelin gene (RELN) increase the risk for schizophrenia and bipolar disorder. We evaluated whether an RELN gene variant, rs362719, which has been associated with increased susceptibility to bipolar disorder, is also associated with susceptibility to schizophrenia. We included 405 Chinese Han schizophrenia patients and 390 controls in our study. The polymorphism was genotyped by PCR and RFLP methods. We found a significant difference in allele frequency distribution (P< 0.05) between schizophrenia patients and controls. The frequency of the A allele was significantly higher in schizophrenia patients than in healthy controls. The effect of SNP rs362719 on allele distribution was significant in female (P < 0.05) but not in male participants (P = 0.473). Besides the gender factor, demographic and clinical characteristics of the rs362719 genotype groups were also analyzed using the chi-square test, but no significant differences were found. We conclude that rs362719 of the RELN gene is associated with susceptibility to schizophrenia in Chinese Han, possibly through a gender-specific mechanism. Further studies will be needed to confirm this genetic risk factor for schizophrenia.

Asunto(s)

Pueblo Asiatico , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Proteínas del Tejido Nervioso/genética , Esquizofrenia/genética , Serina Endopeptidasas/genética , Adulto , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Mutación , Polimorfismo de Nucleótido Simple , Proteína Reelina , Esquizofrenia/etnología

RESUMEN

To look for novel microsatellites in the dystrophin gene for the diagnosis of Duchenne muscular dystrophy, candidate microsatellite sites in the dystrophin gene were analyzed with the SSRHunter software and were also genotyped. Among the 15 candidate microsatellite sites, three novel microsatellite sites in the 60th, 30th, and 2nd intron were found to have a high degree of polymorphism. We submitted these three new loci to the European Molecular Biology Laboratory, under accession Nos. FN547040, FN547041 and FN557526, which were called DXSDMD-in60, DXSDMD-in30 and DXSDMD-in2, respectively. In these three loci, we found 9, 6 and 11 alleles, respectively, in the 205 individuals. In addition, we also detected 20, 19 and 20 genotypes for the three loci in female samples, with a polymorphism information content of more than 0.600. In conclusion, the three microsatellite sites in the intron region of the dystrophin gene have a high degree of polymorphism, and they can be used in population genetics, as well as to provide a theoretical basis for genetic diagnosis and elucidation of molecular mechanisms in Duchenne muscular dystrophy.

Asunto(s)

Distrofina/genética , Repeticiones de Microsatélite , Distrofia Muscular de Duchenne/genética , Polimorfismo Genético , Alelos , Secuencia de Bases , Femenino , Pruebas Genéticas , Genética de Población , Genotipo , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

Brain-derived neurotrophic factor (BDNF) is needed to support neuronal survival and differentiation. It also promotes synaptic remodeling and modulates the function of many other neurotransmitters. BDNF is implicated in major depression (MD), and to a lesser extent, in schizophrenia. In the current study, we examined BDNF polymorphisms (G-712A, C270T and Val66Met) in 202 patients with MD and 323 patients with schizophrenia. Results were compared to 346 healthy individuals. The analysis revealed a strong association between the G-712A genotype distribution and MD (p=0.0005). The frequency of the -712A allele was significantly higher in MD patients than in the healthy controls (p=0.0007). The -712AG heterozygote was associated with higher Hamilton score in MD patients. No association was found between schizophrenia and the three BDNF variants. These findings support an important role of G-712A polymorphism of BDNF in MD, and may guide future studies to identify genetic risk factors for MD.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/genética , Trastorno Depresivo Mayor/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esquizofrenia/genética

RESUMEN

Cardiotrophin-1 (CT-1) is a new member of the interleukin-6 cytokine family that was identified from a mouse embryoid body cDNA library by expression cloning. Mouse CT-1 induces features of hypertrophy in neonatal rat cardiac myocytes and binds to and activates the leukaemia inhibitory factor/gp130 receptor complex. In this work we report the isolation and characterization of cDNA and genomic clones encoding human CT-1. These clones encode a 201 amino acid protein that is 80% identical to the mouse protein. Human CT-1 produced by transfection of the cDNA clones into mammalian cells induces the hypertrophy of neonatal rat cardiac myocytes. Human and mouse CT-1 bind to the leukaemia inhibitory factor receptor on both human and mouse cell lines indicating a lack of species specificity. No binding to the human oncostatin M specific receptor was detected. A 1.7 kb CT-1 mRNA is expressed in adult human heart, skeletal muscle, ovary, colon, prostate and testis and in fetal kidney and lung. The coding region of CT-1 is contained on three exons and is located on human chromosome 16p11.1-16p11.2.

Asunto(s)

Cromosomas Humanos Par 16 , Citocinas/química , Citocinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Secuencia de Consenso , Citocinas/metabolismo , ADN Complementario , Exones , Femenino , Biblioteca de Genes , Células HeLa , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/biosíntesis , Ratas , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transfección

RESUMEN

Thrombopoietin (TPO), the ligand for c-mpl, is a novel cytokine comprising an amino terminal domain with homology to erythropoietin and a glycosylated carboxyl terminal domain that does not bear overall homology to other known proteins. We report the cloning of cDNAs encoding the porcine and murine TPO and the characterization of the human TPO gene. The cDNA for an additional splice form (TPO-2) with a four-amino-acid deletion within the erythropoietin-like domain has been isolated and is conserved between humans, pigs, and mice. Species comparison of TPO shows that the amino terminal erythropoietin-like domain is highly conserved, while the carboxyl terminal domain is less conserved. Recombinant murine TPO and human TPO are each able to activate both the murine and human c-mpl receptors, indicating an absence of strict species specificity. Human TPO is encoded by a single gene consisting of six exons and located on chromosome 3q27-28.

Asunto(s)

Empalme Alternativo , Cromosomas Humanos Par 3 , Proteínas de Neoplasias , Trombopoyetina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Mapeo Cromosómico , Secuencia Conservada , Cartilla de ADN , Eritropoyetina/genética , Exones , Biblioteca Genómica , Humanos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Trombopoyetina , Proteínas Recombinantes/farmacología , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos , Trombopoyetina/biosíntesis , Trombopoyetina/farmacología

RESUMEN

Physiological platelet synthesis is thought to require the humoral activities of meg-CSF and thrombopoietin, which respectively promote proliferation and maturation of megakaryocytic cells. A meg-CSF/thrombopoietin-like protein that is present in plasma of irradiated pigs has been purified and cloned. This protein binds to and activates the c-mpl protein, a member of the cytokine receptor superfamily. The isolated Mpl ligand shares homology with erythropoietin and stimulates both megakaryocytopoiesis and thrombopoiesis.

Asunto(s)

Plaquetas/citología , Megacariocitos/citología , Receptores Inmunológicos/metabolismo , Trombopoyetina/metabolismo , Secuencia de Aminoácidos , Anemia Aplásica/patología , Animales , Secuencia de Bases , Diferenciación Celular , División Celular , Línea Celular , Clonación Molecular , Eritropoyetina/química , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Receptores Inmunológicos/fisiología , Proteínas Recombinantes/biosíntesis , Homología de Secuencia de Aminoácido , Porcinos , Trombopoyetina/química , Trombopoyetina/fisiología , Distribución Tisular

RESUMEN

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.

Asunto(s)

Proteínas Tirosina Quinasas Receptoras/genética , Células 3T3 , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/enzimología , Cromosomas Humanos Par 7 , Clonación Molecular , Cricetinae , ADN Complementario , Humanos , Células Híbridas , Ratones , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas Receptoras/clasificación , Receptor EphB4 , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas

RESUMEN

CDC42, a member of the Rho family of small GTP-binding proteins, regulates cytoskeletal rearrangements required for cell division. Activating mutations in CDC42 that are refractory to GTPase activation confer a phenotype of large, multinucleated cells. Like other small GTP-binding proteins, CDC42 is activated by a guanosine exchange factor and inactivated by a GTPase-activating protein (GAP). An unidentified 25-kDa platelet protein has been shown to function as a specific CDC42GAP. Here we report the cloning of a cDNA encoding this GAP from a human platelet-precursor cell line. Sequence analysis reveals the presence of three consensus box regions characteristic of rhoGAPs. A glutathione S-transferase fusion protein containing the three boxes derived from the new clone strongly stimulated the GTPase activity of CDC42 but was much less effective on other Rho proteins. This indicates that the cDNA clone encodes a specific GAP for CDC42. Sequence analysis also reveals a potential proline-rich Src homology 3 (SH3)-binding domain preceding the first consensus box. Binding experiments show that this motif can interact with the SH3 domains of p85 alpha and of c-Src. Thus, CDC42GAP may function as a link between CDC42 and other signaling pathways.

Asunto(s)

GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Clonación Molecular , ADN Complementario , Activación Enzimática , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa , Humanos , Datos de Secuencia Molecular , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteína de Unión al GTP cdc42

RESUMEN

Heat-stable enterotoxins (STa) are small, cysteine-rich peptides secreted by Escherichia coli that are able to induce diarrhea through the stimulation of an intestine-specific receptor-guanylyl cyclase known as STaR. A 15-amino acid peptide, guanylin, was recently purified from rat jejunum and proposed to be a potential endogenous activator of this receptor. We describe here the cloning and characterization of human and mouse cDNAs encoding precursor proteins of 115 and 116 amino acids, respectively, having guanylin present at their C termini. Expression of the human cDNA in mammalian cells leads to the secretion of proguanylin, an inactive 94-amino acid protein. Guanylin generated by either trypsin or acid treatment of proguanylin was purified and found to bind to, and activate, STaR. Northern blot and in situ hybridization show high-level expression of guanylin mRNA restricted to the intestine, with localization to Paneth cells at the base of the small intestinal crypts. These results demonstrate that guanylin is an endogenous activator of STaR.

Asunto(s)

Hormonas Gastrointestinales , Péptidos/genética , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Expresión Génica , Humanos , Hibridación in Situ , Intestino Delgado/fisiología , Ratones , Datos de Secuencia Molecular , Péptidos Natriuréticos , Sondas de Oligonucleótidos , Especificidad de Órganos , Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

IL-8 is a proinflammatory cytokine that functions as a chemoattractant for neutrophils. Recently, cDNA clones encoding the human neutrophil IL-8R were isolated by an expression cloning strategy. The amino acid sequence of the human IL-8R was sufficiently similar to a published sequence for an isoform of the rabbit FMLP receptor that we considered the possibility that the rabbit sequence might bind IL-8 as well. In order to establish its ligand specificity, we have isolated and characterized cDNA clones encoding the rabbit receptor. These cDNA clones, when expressed in mammalian cells, confer high affinity IL-8 binding (Kd = 3.6 nM), lack detectable binding of FMLP, and produce a transient increase in the intracellular Ca2+ concentration in response to IL-8 but not to FMLP. These data demonstrate that the reported rabbit FMLP receptor is the rabbit IL-8R, not an isoform of the FMLP receptor. In addition, the amino acid sequence of the rabbit IL-8R encoded by these cDNA clones differs at 23 amino acids (of 355) from that previously published.

Asunto(s)

ADN/química , Interleucina-8/metabolismo , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/aislamiento & purificación , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Conejos , Receptores de Formil Péptido , Receptores Inmunológicos/análisis , Receptores de Interleucina-8A

RESUMEN

Interleukin-8 (IL-8) is a member of a family of pro-inflammatory cytokines. Although the best characterized activities of IL-8 include the chemoattraction and activation of neutrophils, other members of this family have a wide range of specific actions including the chemotaxis and activation of monocytes, the selective chemotaxis of memory T cells, the inhibition of hematopoietic stem cell proliferation, and the induction of neutrophil infiltration in vivo. A complementary DNA encoding the IL-8 receptor from human neutrophils has now been isolated. The amino acid sequence shows that the receptor is a member of the superfamily of receptors that couple to guanine nucleotide binding proteins (G proteins). The sequence is 29% identical to that of receptors for the other neutrophil chemoattractants, fMet-Leu-Phe and C5a. Mammalian cells transfected with the IL-8 receptor cDNA clone bind IL-8 with high affinity and respond specifically to IL-8 by transiently mobilizing calcium. The IL-8 receptor may be part of a subfamily of related G protein-coupled receptors that transduce signals for the IL-8 family of pro-inflammatory cytokines.

Asunto(s)

Interleucina-8/metabolismo , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Sondas de ADN , Humanos , Cinética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Plásmidos , ARN Mensajero/genética , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-8A , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Transfección

RESUMEN

The sequence of 20,114 bp of DNA including the human glucose-6-phosphate dehydrogenase (G6PD) gene was determined. The region included a prominent CpG island, starting about 680 nucleotides upstream of the transcription start site, extending about 1050 nucleotides downstream of the start site, and ending just at the start of the first intron. The transcribed region from the start site to the poly(A) addition site covers 15,860 bp. The sequence of the 13 exons agreed with published cDNA sequence and for the 11 exons tested, with the corresponding sequence in a yeast artificial chromosome (YAC). The latter confirms YAC cloning fidelity at the DNA sequence level. Sixteen Alu sequences constitute 24% of the total sequence tract. Four were outside the borders of the mRNA transcript of the gene; all the others were found in a large (9858 bp) intron between exons 2 and 3. Two Alu clusters each contain Alus lying between the monomers of another.

Asunto(s)

Glucosafosfato Deshidrogenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Fúngicos , Clonación Molecular/métodos , Genes , Vectores Genéticos , Humanos , Intrones , Metilación , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/genética

RESUMEN

Vascular endothelial growth factor (VEGF) was purified from media conditioned by bovine pituitary folliculostellate cells (FC). VEGF is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. Complementary DNA clones for bovine and human VEGF were isolated from cDNA libraries prepared from FC and HL60 leukemia cells, respectively. These cDNAs encode hydrophilic proteins with sequences related to those of the A and B chains of platelet-derived growth factor. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins, in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factor. Human 293 cells transfected with an expression vector containing a bovine or human VEGF cDNA insert secrete an endothelial cell mitogen that behaves like native VEGF.

Asunto(s)

Endotelio Vascular/citología , Linfocinas/fisiología , Neovascularización Patológica/fisiopatología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bovinos , División Celular , Clonación Molecular , Biblioteca de Genes , Humanos , Linfocinas/genética , Linfocinas/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.

Asunto(s)

Proteínas Portadoras/genética , Membrana Celular/metabolismo , Etilmaleimida/farmacología , Genes , Aparato de Golgi/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/fisiología , Línea Celular , Citosol/metabolismo , Genes Fúngicos , Cinética , Datos de Secuencia Molecular , Proteínas Sensibles a N-Etilmaleimida , Saccharomyces cerevisiae/metabolismo

RESUMEN

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide, and on various other cells as a receptor for atrial natriuretic peptides. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphorylation. Unexpectedly, the enzyme is homologous to the protein kinase family.

Asunto(s)

Guanilato Ciclasa , Proteínas Quinasas , Receptores de Superficie Celular , Espermatozoides/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/enzimología , ADN/genética , ADN Recombinante , Glicosilación , Guanilato Ciclasa/genética , Guanilato Ciclasa/aislamiento & purificación , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Erizos de Mar , Homología de Secuencia de Ácido Nucleico

RESUMEN

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.

Asunto(s)

Clonación Molecular , Metaloendopeptidasas/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/enzimología , ADN/análisis , Humanos , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Neprilisina , Conejos , Ratas