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1.
Mol Genet Metab ; 102(1): 61-8, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20884265

RESUMEN

Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells (AdeI) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the "signature sequence" of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency.


Asunto(s)
Adenilosuccinato Liasa/genética , Mutación Missense , Adenilosuccinato Liasa/biosíntesis , Adenilosuccinato Liasa/deficiencia , Sustitución de Aminoácidos , Animales , Trastorno Autístico , Células CHO , Dominio Catalítico , Cricetinae , Cricetulus , Pruebas de Enzimas , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Errores Innatos del Metabolismo de la Purina-Pirimidina/enzimología , ARN Mensajero/química , Transcripción Genética
2.
Protein Expr Purif ; 51(2): 227-34, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16973378

RESUMEN

The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel-nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly alpha-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 degrees C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase.


Asunto(s)
Adenilosuccinato Liasa/biosíntesis , Adenilosuccinato Liasa/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/metabolismo , Dicroismo Circular , Clonación Molecular , Activación Enzimática , Estabilidad de Enzimas , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Compuestos de Sulfhidrilo/análisis , Temperatura
3.
Hum Mol Genet ; 9(10): 1501-13, 2000 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-10888601

RESUMEN

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits. Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity. We studied the disorder at various levels in a group of six patients with ADSL deficiency. We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping. Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant. Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level. The results showed only the unspliced ADSL to be active. The gene consists of 13 exons spanning 23 kb. The promotor region shows typical features of the housekeeping gene. Eight mutations were identified in a group of six patients. The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype. All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates. However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients. This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.


Asunto(s)
Adenilosuccinato Liasa/química , Adenilosuccinato Liasa/deficiencia , Adenilosuccinato Liasa/biosíntesis , Adenilosuccinato Liasa/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Niño , Preescolar , Clonación Molecular , ADN Complementario/metabolismo , Escherichia coli/metabolismo , Exones , Femenino , Fibroblastos/metabolismo , Genotipo , Humanos , Lactante , Cinética , Masculino , Datos de Secuencia Molecular , Mutación , Fenotipo , Regiones Promotoras Genéticas , Isoformas de Proteínas , Análisis de Secuencia de ADN , Temperatura , Distribución Tisular
4.
Eur J Biochem ; 234(3): 843-8, 1995 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-8575443

RESUMEN

To investigate the working hypotheses that stem cells or their early descendants are prime targets for neoplastic transformation, and that the degree to which a neoplasm retains the immature phenotype is an important determinant of tumor aggressiveness, we have identified several mRNAs that are downregulated during the in vitro differentiation of HT29-D4 colon carcinoma cells. These genes include heat-shock cognate protein Hsc70, adenylosuccinate lyase, B23/nucleophosmin, alpha-tubulin, and a novel gene designated DS-1. The DS-1 mRNA has a length of approximately 0.9 kb and is downregulated 4.7-fold upon differentiation. From the DS-1 cDNA, a protein of 206 amino acids with a molecular mass of 24 kDa and an isoelectric point of 10.9 can be deduced. An antiserum directed against a synthetic peptide detected a minor band of the expected size in Western blots, as well as a major band of lower size that may represent a processed form of the protein.


Asunto(s)
Diferenciación Celular/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , ARN Mensajero/genética , Adenilosuccinato Liasa/biosíntesis , Adenilosuccinato Liasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Neoplasias del Colon/patología , Cartilla de ADN , ADN Complementario , Regulación hacia Abajo , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Nucleofosmina , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas , Tubulina (Proteína)/biosíntesis , Tubulina (Proteína)/genética , Células Tumorales Cultivadas
5.
J Biol Chem ; 268(26): 19710-6, 1993 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-8366112

RESUMEN

Adenylosuccinate adenosine 5'-monophosphate lyase (EC 4.3.2.2; ASL) catalyzes two distinct reactions in adenosine 5'-monophosphate (AMP) biosynthesis. A S413P mutation in ASL segregates with mental retardation in an affected family (Stone, R. L., Aimi, J., Barshop, B. A., Jaeken, J., Van den Berghe, G., Zalkin, H., and Dixon, J. E. (1992) Nature Genet. 1, 59-63). ASL and S413P ASL have been expressed, purified, and kinetically characterized. Lowering the Escherichia coli growth temperature to 25 degrees C and the concentration of inducer, isopropyl-1-thio-beta,D-galactopyranoside, to 40 microM was necessary for synthesis of soluble, tetrameric enzymes. The recombinant enzymes were purified to homogeneity using anion exchange chromatography followed by chromatography on Blue 2A Sepharose. At pH 7.0 and 25 degrees C, the kcat for cleavage of 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAI-CAR) by ASL was 90 s-1 with a Km of 2.35 microM. The kcat for adenylosuccinate (SAMP) cleavage was 97 s-1 with a Km of 1.79 microM. The catalytic mechanism involved one general base catalyst (pK alpha = 6.4) and one general acid catalyst (pK alpha = 7.5). ASL follows an ordered uni-bi reaction mechanism with fumarate released first. 5-Amino-4-imidazolecarboxamide ribotide (AICAR) and AMP were competitive with SAICAR and SAMP (Ki[AICAR] = 11.3 microM; Ki[AMP] = 9.2 microM), whereas fumarate inhibited noncompetitively (Kii = 2.3 mM, Kis = 2.8 mM). The competitive inhibition by AICAR and AMP suggests a single active site that binds both SAICAR and SAMP. The kinetic constants at pH 7.0, 25 degrees C and the kcat/Km versus pH profiles for ASL and S413P ASL were very similar. These results are consistent with S413P being a structural rather than a catalytic defect.


Asunto(s)
Adenilosuccinato Liasa/metabolismo , Adenilosuccinato Liasa/biosíntesis , Adenilosuccinato Liasa/aislamiento & purificación , Secuencia de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Secuencia de Bases , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Plásmidos , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleótidos/metabolismo , Especificidad por Sustrato
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