RESUMEN
Adult subjects with classical phenylketonuria (PKU) who were diagnosed and treated neonatally participated in this long-term follow-up study. Twenty-four subjects received neuropsychological (NP) assessment and a subset received magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) to identify: (1) pattern of cognitive dysfunction; (2) effect of high blood phenylalanine (Phe) level at time of cognitive testing; and (3) treatment variables that may be associated with cognitive difficulties in adulthood. All subjects had average IQ except one subject in the borderline range. Diet was initiated by the 15th day of life. All subjects except one were on diet until age 6 years (mean years of treatment = 15). Blood Phe levels at cognitive testing ranged from 157 to 1713 micromol/L (mean = 1038); 11 subjects had levels < 1000 micromol/L and 13 subjects had levels >1000 micromol/L. Results suggest that adults with early-treated PKU demonstrate specific cognitive deficits, a number of which are associated with the frontal and temporal area of the brain. Deficits were noted in several domains including executive functioning, attention, verbal memory, expressive naming and verbal fluency. Self-report measures of depression and anxiety were generally in the normal/mild range. The group with a Phe level > 1000 micromol/L scored lower than the group with Phe level < 1000 micromol/L on measures of focused attention, verbal fluency, reaction time, verbal recognition memory, visual memory and naming. Tests of cognitive functioning were often correlated with measures of treatment during childhood rather than with Phe level at the time of cognitive testing. Subjects with abnormal MRI scored significantly lower on two cognitive tests (Trails A and CVLT Recognition Memory). We found no significant correlation between current brain Phe level obtained through MRS (n = 10) and neuropsychological functioning. Future longitudinal investigation with a larger sample size will assist in clarifying the aetiology of neuropsychological deficits and association with treatment history.
Asunto(s)
Fenilcetonurias/patología , Fenilcetonurias/psicología , Adulto , Encéfalo/patología , Trastornos del Conocimiento/diagnóstico , Femenino , Lóbulo Frontal/patología , Humanos , Inteligencia , Pruebas de Inteligencia , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Pruebas Neuropsicológicas , Fenilalanina/sangre , Lóbulo Temporal/patologíaRESUMEN
During 1967-1983, the Maternal and Child Health Division of the Public Health Services funded a collaborative study of 211 newborn infants identified on newborn screening as having phenylketonuria (PKU). Subsequently, financial support was provided by the National Institute of Child Health and Human Development (NICHD). The infants were treated with a phenylalanine (Phe)-restricted diet to age 6 years and then randomized either to continue the diet or to discontinue dietary treatment altogether. One hundred and twenty-five of the 211 children were then followed until 10 years of age. In 1998, NICHD scheduled a Consensus Development Conference on Phenylketonuria and initiated a study to follow up the participants from the original Collaborative Study to evaluate their present medical, nutritional, psychological, and socioeconomic status. Fourteen of the original clinics (1967-1983) participated in the Follow-up Study effort. Each clinic director was provided with a list of PKU subjects who had completed the original study (1967-1983), and was asked to evaluate as many as possible using a uniform protocol and data collection forms. In a subset of cases, magnetic resonance imaging and spectroscopy (MRI/MRS) were performed to study brain Phe concentrations. The medical evaluations revealed that the subjects who maintained a phenylalanine-restricted diet reported fewer problems than the diet discontinuers, who had an increased rate of eczema, asthma, mental disorders, headache, hyperactivity and hypoactivity. Psychological data showed that lower intellectual and achievement test scores were associated with dietary discontinuation and with higher childhood and adult blood Phe concentrations. Abnormal MRI results were associated with higher brain Phe concentrations. Early dietary discontinuation for subjects with PKU is associated with poorer outcomes not only in intellectual ability, but also in achievement test scores and increased rates of medical and behavioural problems.
Asunto(s)
Fenilcetonurias , Adulto , Química Encefálica , Niño , Continuidad de la Atención al Paciente , Escolaridad , Estudios de Seguimiento , Humanos , Imagen por Resonancia Magnética , Fenilalanina/administración & dosificación , Fenilalanina/análisis , Fenilalanina/sangre , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/complicaciones , Fenilcetonurias/diagnóstico , Fenilcetonurias/dietoterapia , Fenilcetonurias/psicología , Análisis de Regresión , Clase Social , Escalas de WechslerRESUMEN
In this report, a novel positive-negative epitope tagging approach was developed to study the cellular processing of beta amyloid precursor protein (beta APP). Amino acids centered around the alpha-secretase cleavage site within the A beta sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant beta APP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled beta APP by beta- and gamma-secretase(s) results in the release of an epitope-tagged A beta peptide, whereas cleavage by alpha-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled beta APP via the amyloidogenic pathway. Secretion of epitope-tagged A beta was prevented by MDL 28170, a previously described gamma-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged beta APP was not different from wild-type beta APP. These results suggest that positive-negative epitope-tagged beta APP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer's disease.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Epítopos/metabolismo , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Anticuerpos Monoclonales/inmunología , Ácido Aspártico Endopeptidasas , Western Blotting , Línea Celular , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Dipéptidos/farmacología , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunohistoquímica , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Inhibidores de Proteasas/análisis , Inhibidores de Proteasas/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Sensibilidad y Especificidad , TransfecciónRESUMEN
We report the discovery of a class of pyrazole-based compounds that are potent inhibitors of the dihydroorotate dehydrogenase of Helicobacter pylori but that do not inhibit the cognate enzymes from Gram-positive bacteria or humans. In culture these compounds inhibit the growth of H. pylori selectively, showing no effect on other Gram-negative or Gram-positive bacteria or human cell lines. These compounds represent the first examples of H. pylori-specific antibacterial agents. Cellular activity within this structural class appears to be due to dihydroorotate dehydrogenase inhibition. Minor structural changes that abrogate in vitro inhibition of the enzyme likewise eliminate cellular activity. Furthermore, the minimum inhibitory concentrations of these compounds increase upon addition of orotate to the culture medium in a concentration-dependent manner, consistent with dihydroorotate dehydrogenase inhibition as the mechanism of cellular inhibition. The data presented here suggest that targeted inhibition of de novo pyrimidine biosynthesis may be a valuable mechanism for the development of antimicrobial agents selective for H. pylori.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Helicobacter pylori/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/antagonistas & inhibidores , Pirimidinas/biosíntesis , Secuencia de Aminoácidos , Dihidroorotato Deshidrogenasa , Relación Dosis-Respuesta a Droga , Helicobacter pylori/enzimología , Cinética , Datos de Secuencia Molecular , Oxidorreductasas/química , Ubiquinona/química , Ubiquinona/metabolismoRESUMEN
Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.
Asunto(s)
Endopeptidasas/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Membrana Celular/metabolismo , Endopeptidasas/metabolismo , Pruebas de Precipitina , Presenilina-1 , Presenilina-2 , Especificidad por SustratoRESUMEN
Dihydroorotate dehydrogenase is a critical enzyme of de novo pyrimidine biosynthesis in prokaryotic and eukaryotic cells. Differences in the primary structure of the enzymes from Gram-positive and -negative bacteria and from mammals indicate significant structural divergence among these enzymes. We have identified a class of small molecules, the thiadiazolidinediones, that inhibit prototypical enzymes from Gram-positive and -negative bacteria, but are inactive against the human enzyme. The most potent compound in our collection functioned as a time-dependent irreversible inactivator of the bacterial enzymes with k(inact)/K(i) values of 48 and 500 M(-1) sec(-1) for the enzymes from Escherichia coli and Enterococcus faecalis, respectively. The data presented here indicate that it is possible to inhibit prokaryotic dihydroorotate dehydrogenases selectively while sparing the mammalian enzyme. Thus, this enzyme may represent a valuable target for the development of novel antibiotic compounds.
Asunto(s)
Antibacterianos/farmacología , Enterococcus faecalis/enzimología , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/antagonistas & inhibidores , Tiadiazoles/farmacología , Dihidroorotato Deshidrogenasa , Enterococcus faecalis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Cinética , Pruebas de Sensibilidad MicrobianaRESUMEN
We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor XIIIa/antagonistas & inhibidores , Factor XIIIa/inmunología , Fibrina/metabolismo , Imidazoles/farmacología , Inmunoglobulina G/farmacología , Reología/efectos de los fármacos , Fenómenos Biomecánicos , Fibrina/química , Fibrinógeno/metabolismo , Humanos , Imidazoles/síntesis química , Inmunoglobulina G/inmunologíaRESUMEN
CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.
Asunto(s)
Dióxido de Carbono , Oxigenasas de Función Mixta/análisis , Cinética , Conteo por Cintilación , Sensibilidad y EspecificidadAsunto(s)
Emigrantes e Inmigrantes , Instituciones de Salud , Política de Salud , Salud Pública , Relaciones Raciales , Emigrantes e Inmigrantes/educación , Emigrantes e Inmigrantes/historia , Emigrantes e Inmigrantes/legislación & jurisprudencia , Emigrantes e Inmigrantes/psicología , Emigración e Inmigración/historia , Emigración e Inmigración/legislación & jurisprudencia , Instituciones de Salud/economía , Instituciones de Salud/historia , Instituciones de Salud/legislación & jurisprudencia , Política de Salud/economía , Política de Salud/historia , Política de Salud/legislación & jurisprudencia , Historia del Siglo XX , Humanos , México/etnología , Salud Pública/economía , Salud Pública/educación , Salud Pública/historia , Salud Pública/legislación & jurisprudencia , Relaciones Raciales/historia , Relaciones Raciales/legislación & jurisprudencia , Relaciones Raciales/psicología , Grupos Raciales/educación , Grupos Raciales/etnología , Grupos Raciales/historia , Grupos Raciales/legislación & jurisprudencia , Grupos Raciales/psicología , Texas/etnología , Migrantes/educación , Migrantes/historia , Migrantes/legislación & jurisprudencia , Migrantes/psicología , Estados Unidos/etnologíaAsunto(s)
Eugenesia , Jerarquia Social , Madres , Médicos , Bienestar Social , Salud de la Mujer , Eugenesia/historia , Eugenesia/legislación & jurisprudencia , Femenino , Jerarquia Social/historia , Historia del Siglo XX , Humanos , Lactante , Cuidado del Lactante/economía , Cuidado del Lactante/historia , Cuidado del Lactante/legislación & jurisprudencia , Cuidado del Lactante/psicología , Bienestar del Lactante/economía , Bienestar del Lactante/etnología , Bienestar del Lactante/historia , Bienestar del Lactante/legislación & jurisprudencia , Bienestar del Lactante/psicología , Recién Nacido , México/etnología , Madres/educación , Madres/historia , Madres/legislación & jurisprudencia , Madres/psicología , Médicos/economía , Médicos/historia , Médicos/legislación & jurisprudencia , Médicos/psicología , Sistemas Políticos/historia , Embarazo , Reproducción , Derechos Sexuales y Reproductivos/economía , Derechos Sexuales y Reproductivos/educación , Derechos Sexuales y Reproductivos/historia , Derechos Sexuales y Reproductivos/legislación & jurisprudencia , Derechos Sexuales y Reproductivos/psicología , Bienestar Social/economía , Bienestar Social/etnología , Bienestar Social/historia , Bienestar Social/legislación & jurisprudencia , Bienestar Social/psicología , Mujeres/educación , Mujeres/historia , Mujeres/psicología , Salud de la Mujer/etnología , Salud de la Mujer/historia , Derechos de la Mujer/economía , Derechos de la Mujer/educación , Derechos de la Mujer/historia , Derechos de la Mujer/legislación & jurisprudenciaRESUMEN
In previous chemical modification studies on bovine aspartyl (asparaginyl) beta-hydroxylase, cysteines were implicated as critical catalytic residues. Using site-directed mutagenesis, the five cysteine residues located in a highly conserved region of the enzyme identified as the active site were individually mutated to alanine. Substitutions at cysteine 637, 644, 656, 681, and 696 resulted in active mutant enzymes indicating that these residues are not required for catalysis.
Asunto(s)
Cisteína/química , Oxigenasas de Función Mixta/metabolismo , Alanina/genética , Animales , Sitios de Unión/genética , Catálisis , Bovinos , Mutagénesis Sitio-Dirigida/genética , Proteínas Recombinantes/metabolismoRESUMEN
Post-translational modifications by transglutaminase may contribute to the remodeling of cellular architecture in the development of lens fiber cells, and there is evidence that the enzyme may also play a role in cataract formation. It catalyses hydrolytic deamidations as well as amide exchanges on select glutamine side chains at endo positions in a small subset of proteins of the lens. N epsilon(gamma-glutamyl)lysine crosslinks, the characteristic hallmarks of transglutaminase activity, were identified in polymers isolated from human cataract. Following up on our earlier studies relating to the inhibition of protein crosslinking by the Ca(2+)-activated transglutaminase in the lens, we have now examined the effects of 2-[(2-oxopropyl)thio]-imidazolium derivatives, recently described as active site-directed inhibitors for this family of enzymes. First, we have shown that the compounds at concentrations of 1-2 microM were effective in blocking the transamidating activities of partially purified lens transglutaminase. Then we focused on their efficacy in preventing the formation of the ca. 55 kDa beta crystallin dimers in the whole lens tissue. The production of these dimers, crosslinked by N epsilon(gamma-glutamyl)lysine isopeptide bridges, is an early sign of transglutaminase action in rabbit lens, and it can be readily documented by the SDS-PAGE analysis of proteins remaining in the soluble phase after brief exposure of the homogenate to Ca2+. The new compounds proved to be potent inhibitors of transglutaminase also in this preparation, preventing the crosslinking event at ca. 1 microM concentration. Moreover, even when applied at a 1,000-fold greater concentration (2 mM), they did not interfere with the action of calpain which, similarly to the activation of the transglutaminase system, is triggered by the addition of Ca2+. The high selectivity of the new compounds for differentially blocking only the transglutaminase and not the calpain of the lens, is all the more remarkable because these two enzymes share several mechanistic and structural similarities.
Asunto(s)
Cristalinas/metabolismo , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Cristalino/enzimología , Transglutaminasas/antagonistas & inhibidores , Animales , Calpaína/antagonistas & inhibidores , Reactivos de Enlaces Cruzados/metabolismo , ConejosRESUMEN
To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.
Asunto(s)
Antígenos de Superficie/genética , Carcinoma Hepatocelular/enzimología , Colangiocarcinoma/enzimología , Oxigenasas de Función Mixta/genética , Adulto , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/inmunología , Carcinoma Hepatocelular/genética , Línea Celular Transformada , Colangiocarcinoma/genética , Clonación Molecular , Neoplasias del Colon/inmunología , Biblioteca de Genes , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
To gain insight into the role of the eukaryotic translation initiation factor, eIF-5A, we investigated the subcellular distribution of this protein in several cultured cell types and at different stages of the cell cycle using a highly potent monospecific polyclonal antibody to eIF-5A. Studies using indirect immunofluorescence and confocal microscopy in conjunction with subcellular fractionation demonstrate that eIF-5A is primarily localized in the cytoplasm of cells. This cytoplasmic location of eIF-5A is not significantly altered in different stages of the cell cycle and the subcellular distribution pattern of eIF-5A is not changed by viral oncogene transformation. Cell fractionation experiments identified two populations of eIF-5A in the cytoplasm, a soluble fraction and a fraction bound to internal membranes. By double immunofluorescence staining with an antibody against calnexin, a resident protein of the endoplasmic reticulum (ER), we demonstrate that the membrane-bound fraction of eIF-5A colocalizes with the ER and not with the cytoskeleton. Expression of Rev, a regulatory protein of human immunodeficiency virus type 1 (HIV-1), does not alter the subcellular distribution of endogenous eIF-5A in these cells. eIF-5A is detected in all tissues and cells examined including extracts prepared from Xenopus oocytes. Our results indicate that eIF-5A is a ubiquitous cytoplasmic protein and suggest that a site of eIF-5A function is likely to be in association with the ER.
Asunto(s)
Células 3T3/ultraestructura , Síndrome de Inmunodeficiencia Adquirida/virología , VIH-1/genética , Factores de Iniciación de Péptidos/análisis , Proteínas de Unión al ARN , Fracciones Subcelulares/química , Células 3T3/química , Animales , Especificidad de Anticuerpos , Western Blotting , Ciclo Celular/fisiología , Citoesqueleto/química , Retículo Endoplásmico/química , Expresión Génica/fisiología , Productos del Gen rev/genética , Ratones , Microscopía Confocal , Factores de Iniciación de Péptidos/inmunología , Biosíntesis de Proteínas/fisiología , Proteínas Virales de Fusión/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
The roles in catalysis of several residues in bovine aspartyl (asparaginyl) beta-hydroxylase that are located in a region of homology among alpha-ketoglutarate-dependent dioxygenases were investigated using site-directed mutagenesis. Previous studies have shown that when histidine 675, an invariant residue located in this highly conserved region, was mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has been undertaken to define the catalytic role of this essential residue. The partial hydroxylase activity observed with some amino acid replacements for histidine 675 correlates with the potential to coordinate metals and not with size, charge, or hydrophobic character. Furthermore, the increase in Km for Fe2+ observed with the H675D and H675E mutant enzymes can account for their partial activities relative to wild type. No significant changes in the Km for alpha-ketoglutarate (at saturating Fe2+) or Vmax were observed for these mutants. These results support the conclusion that histidine 675 is specifically involved in Fe2+ coordination. Further site-directed mutagenesis of other highly conserved residues in the vicinity of position 675 demonstrates the importance of this region of homology in catalysis for Asp (Asn) beta-hydroxylase and, by analogy, other alpha-ketoglutarate-dependent dioxygenases.
Asunto(s)
Histidina , Hierro/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Pollos , Secuencia Conservada , Humanos , Cinética , Oxigenasas de Función Mixta/biosíntesis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
N1-guanyl-1,7-diaminoheptane (GC7) is a potent inhibitor of deoxyhypusine synthase (DHS), the enzyme that catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF-5A). Since eIF-5A is the only known cellular substrate for DHS and GC7 has been reported to block the proliferation of CHO cells, it has been suggested that DHS may be a novel target for anti-cancer therapy. In the present study we investigated the antiproliferative effect of GC7 on several tumorigenic cell lines under various growth conditions. We found that this compound inhibits the proliferation of H9 cells in suspension culture and the growth of HeLa cells and v-src-transformed NIH3T3 cells under both anchorage-dependent and anchorage-independent conditions. Moreover, studies with NIH3T3 cells and v-src-transformed NIH3T3 cells show that GC7 inhibits the growth of both cell lines in monolayer culture with similar potency and could not reverse the transformed phenotype. In addition, the v-src-transformed cells grown under both anchorage-dependent and anchorage-independent conditions showed similar sensitivity toward GC7. These data indicate that GC7 acts as a general antiproliferative agent and does not appear to preferentially target tumorigenic cell types. Cell cycle analysis show that GC7 reduces the CHO-K1 cell population in the G1-phase of the cell cycle by 42% and increases the number of cells in the S-phase by 44%. This cell cycle distribution profile strikingly resembles the distribution of cells treated with puromycin. This result supports the hypothesis that the synthesis of a subset of proteins important for the S-phase progression of CHO-K1 cells might be dependent upon hypusinated eIF-5A. Thus the antiproliferative effect of GC7 appears to be related to its interference with the progression of cell cycle, which also provides a possible explanation for the lack of selectivity of GC7 between nontransformed and transformed cell types tested in this study.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Proteínas de Unión al ARN , Células 3T3/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada/efectos de los fármacos , Guanidinas/farmacología , Guanina/farmacología , Células HeLa/efectos de los fármacos , Humanos , Ratones , Factores de Iniciación de Péptidos/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.
Asunto(s)
Eritrocitos/enzimología , Imidazoles/farmacología , Transglutaminasas/antagonistas & inhibidores , Animales , Sitios de Unión , Cisteína/química , Perros , Glutatión/química , Humanos , Concentración de Iones de Hidrógeno , Yodoacetamida/farmacología , Cinética , Espectroscopía de Resonancia Magnética , Peso Molecular , Compuestos de Sulfhidrilo/químicaRESUMEN
The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC 1.14.11.16) specifically hydroxylates one aspartic or asparagine residue in certain epidermal growth factor-like domains of a number of proteins. The expression in Escherichia coli, purification, characterization of a fully active catalytic domain, and evidence for the identification of an active-site region of this enzyme are described. Sequence alignment analyses among the vertebrate alpha-ketoglutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant aspartyl (asparaginyl) beta-hydroxylase involved in substrate binding and/or catalysis. Based upon these studies, an alignment of the C-terminal regions of prolyl and lysyl hydroxylase and of aspartyl (asparaginyl) beta-hydroxylase is proposed. When histidine-675, an invariant residue located in a region of homology within this alignment, was mutated to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H675A), no enzymatic activity was detected. Chemical modification studies show that the wild-type protein is protected from iodo[14C]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Bovinos , Clonación Molecular , Escherichia coli , Humanos , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , VertebradosRESUMEN
A neutral proteolytic activity that converts human Big endothelin-1 (Big Et-1) to endothelin-1 has been identified from a human endothelial hybrid cell line, EAHY 926. This enzyme is an integral membrane protein and cofractionates with other enzymes typically found in the plasma membrane. The activity has been solubilized with nonionic detergents and purified 1000-fold by a combination of lectin affinity chromatography, ion-exchange chromatography, and chromatography on red-dye agarose. The partially purified activity is a metalloenzyme based upon its sensitivity to chelating agents, competitive inhibition by phosphoramidon, and reconstitution with ZnCl2 or CoCl2 following EDTA inactivation. The enzyme appears to be unique, however, as it is not inhibited by specific inhibitors of known metalloproteases. It correctly processes Big Et-1 to Et-1 and the complementary C-terminal fragment with a sharp pH optimum near 7.0. Both the Km for Big Et-1 and the Ki for phosphoramidon are pH-dependent, with values of 5-7 and 3.5 microM, respectively, at pH 7.0. The enzyme also cleaves Big Et-2 with a Km of 27.9 microM and a Vmax one-third that for Big Et-1 but has no appreciable activity toward Big Et-3. An s20,w of 9.5 S was determined by sucrose density ultracentrifugation in H2O and D2O. When combined with a Stokes radius of 56 A determined by gel filtration, the enzyme had a calculated apparent molecular weight of 250,000. Conditions have been established to renature the activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions activity was detected in a protein band at 280 kDa, in agreement with the aforementioned molecular weight determination. From these results, a kcat/Km of 1 x 10(6) M-1 s-1 was estimated for the purified enzyme with Big Et-1 as a substrate, which is a reasonable value for a protease acting upon its physiologic substrate. Several criteria indicate that the activity isolated from EAHY cells is the physiologically relevant endothelial-derived endothelin converting enzyme. On the basis of our results, this enzyme is present in low abundance in endothelial cells and at least a 100,000-fold purification will be required to obtain a homogeneous preparation. However, because EAHY cells can be grown in large numbers, they can supply the quantities of enzyme required both for biochemical studies and for the development of specific inhibitors.