RESUMO
Dentinogenesis imperfecta (DI) is the main orodental manifestation of osteogenesis imperfecta (OI) caused by COL1A1 or COL1A2 heterozygous pathogenic variants. Its prevalence varies according to the studied population. Here, we report the molecular analysis of 81 patients with OI followed at reference centers in Brazil and France presenting COL1A1 or COL1A2 variants. Patients were submitted to clinical and radiographic dental examinations to diagnose the presence of DI. In addition, a systematic literature search and a descriptive statistical analysis were performed to investigate OI/DI phenotype-genotype correlation in a worldwide sample. In our cohort, 50 patients had COL1A1 pathogenic variants, and 31 patients had COL1A2 variants. A total of 25 novel variants were identified. Overall, data from a total of 906 individuals with OI were assessed. Results show that DI was more frequent in severe and moderate OI cases. DI prevalence was also more often associated with COL1A2 (67.6%) than with COL1A1 variants (45.4%) because COL1A2 variants mainly lead to qualitative defects that predispose to DI more than quantitative defects. For the first time, 4 DI hotspots were identified. In addition, we showed that 1) glycine substitution by branched and charged amino acids in the α2(I) chain and 2) substitutions occurring in major ligand binding regions-MLRB2 in α1(I) and MLBR 3 in α2(I)-could significantly predict DI (P < 0.05). The accumulated variant data analysis in this study provides a further basis for increasing our comprehension to better predict the occurrence and severity of DI and appropriate OI patient management.
Assuntos
Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo I , Dentinogênese Imperfeita , Osteogênese Imperfeita , Humanos , Colágeno Tipo I/genética , Dentinogênese Imperfeita/genética , Estudos de Associação Genética , Mutação , Osteogênese Imperfeita/diagnóstico por imagem , Osteogênese Imperfeita/genéticaRESUMO
Hereditary xanthinuria is classified into three categories. Classical xanthinuria type I lacks only xanthine dehydrogenase activity, while type II and molybdenum cofactor deficiency also lack one or two additional enzyme activities. In the present study, we examined four individuals with classical xanthinuria to discover the cause of the enzyme deficiency at the molecular level. One subject had a C to T base substitution at nucleotide 682 that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 228. The duodenal mucosa from the subject had no xanthine dehydrogenase protein while the mRNA level was not reduced. The two subjects who were siblings with type I xanthinuria were homozygous concerning this mutation, while another subject was found to contain the same mutation in a heterozygous state. The last subject who was also with type I xanthinuria had a deletion of C at nucleotide 2567 in cDNA that should generate a termination codon from nucleotide 2783. This subject was homozygous for the mutation and the level of mRNA in the duodenal mucosa from the subject was not reduced. Thus, in three subjects with type I xanthinuria, the primary genetic defects were confirmed to be in the xanthine dehydrogenase gene.
Assuntos
Coenzimas , Mutação Puntual , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Deleção de Sequência , Xantina Desidrogenase/genética , Xantinas/urina , Adulto , Idoso , Códon , Primers do DNA , Duodeno , Humanos , Mucosa Intestinal/enzimologia , Masculino , Metaloproteínas/metabolismo , Molibdênio/metabolismo , Cofatores de Molibdênio , Reação em Cadeia da Polimerase , Pteridinas/metabolismo , Erros Inatos do Metabolismo da Purina-Pirimidina/enzimologia , Erros Inatos do Metabolismo da Purina-Pirimidina/urina , RNA Mensageiro/metabolismo , Xantina , Xantina Desidrogenase/deficiênciaRESUMO
The present detailed analysis of the sequence of the extremely long (967 bp) 5'-noncoding region of a rat brain argininosuccinate lyase cDNA clone, reveals several features of interest. Multiple copies of partial and inverted (antisense) B1 repeats and multiple upstream ATG codons are present in the region, which suggests a possibility of translational control of the argininosuccinate lyase gene expression in rat brain.
Assuntos
Argininossuccinato Liase/genética , Encéfalo/enzimologia , RNA Mensageiro/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon , Dados de Sequência Molecular , Família Multigênica , Biossíntese de Proteínas , RatosRESUMO
The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined. The nucleotide sequence contains an open reading frame of 1191 base pairs and encodes an amino acid sequence of 397 residues which exhibits 86.6% homology with that of the rat enzyme. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblasts and intercostal muscle.
Assuntos
Acetil-CoA C-Aciltransferase/genética , DNA Complementar/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Humanos , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/análise , Homologia de Sequência de AminoácidosRESUMO
The primary structure of human xanthine dehydrogenase (hXDH) was determined by cloning and sequence analysis of the cDNAs encoding the enzyme. The nucleotide (nt) sequence has an open reading frame of 3999 nt encoding a protein of 1333 amino acids (aa) with a calculated M(r) of 146,604. The deduced aa sequence of hXDH is homologous to the previously reported rat XDH (rXDH) and Drosophila melanogaster XDH sequences with identities of 90.2 and 52.0%, respectively. The aa residues involved in both the reversible and the irreversible conversion from the dehydrogenase type to the oxidase type of rXDH are completely conserved between the rat and the human enzymes. This implies that the molecular mechanisms of the conversion of hXDH from dehydrogenase to oxidase are common to those of the well-characterized rXDH. Five sequence variations were detected in the isolated cDNA clones. Spot blot hybridization using flow-sorted human chromosome revealed that the hXDH-encoding gene (hXDH) was located on chromosome 2.
Assuntos
Cromossomos Humanos Par 2 , Xantina Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA , Drosophila melanogaster , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Mapeamento por Restrição , Xantina Desidrogenase/química , Xantina Desidrogenase/metabolismo , Xantina Oxidase/genética , Xantina Oxidase/metabolismoRESUMO
The function of the SRH1 product, the yeast homologue of the 54 kDa subunit of the mammalian signal recognition particle, has been analyzed using a galactose dependent mutant of the gene. SRH1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SRH1 copy disrupted. This mutant grows normally on galactose medium but slows down the growth about 10 h after transfer to glucose medium. At the same time, precursor forms of secretory proteins, alpha-mating factor and invertase, accumulate in the cells. This result indicates that the SRH1 product is involved in translocation of precursors of secretory proteins across the endoplasmic reticulum membrane in yeast cells.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Proteínas Fúngicas/metabolismo , Galactose/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Substâncias Macromoleculares , Fator de Acasalamento , Dados de Sequência Molecular , Peso Molecular , Sondas de Oligonucleotídeos , Peptídeos/genética , Peptídeos/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Precursores de Proteínas/genética , Mapeamento por Restrição , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido Nucleico , Partícula de Reconhecimento de Sinal , beta-FrutofuranosidaseRESUMO
In the present study, we isolated clones of human argininosuccinate lyase (ASL) cDNA from a liver cDNA library using a clone of rat ASL cDNA and analyzed human ASL cDNA nucleotide sequence. The results reveal that the sequence of human ASL cDNA published by O'Brien et al. in 1986 [Proc. Natl. Acad. Sci USA 83, 7211-7215] had one-base deletions at three independent positions in the coding regions near the COOH-terminus, which caused frame-shift variations in the amino acid sequence. Amino acid sequencing of peptides prepared from purified human liver ASL showed our predicted amino acid sequence to be correct.
Assuntos
Argininossuccinato Liase/genética , DNA/isolamento & purificação , Liases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Genes , Humanos , Fígado/enzimologia , Dados de Sequência Molecular , RatosRESUMO
Argininosuccinate lyase (EC 4.3.2.1) is an enzyme present in the brain of ureotelic animals. Using as a probe rat liver argininosuccinate lyase cDNA, already isolated and sequenced (Amaya, Y., Matsubasa, T., Takiguchi, M., Kobayashi, K., Saheki, T., Kawamoto, S. and Mori, M., J. Biochem., 103 (1988) 177-181), we screened a rat brain cDNA library constructed in the lambda gt11 expression vector and obtained a single cDNA clone. This cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,390), a 5'-untranslated sequence of 967 bp and a 3'-untranslated sequence of 74 bp. The length of the 5'-non-coding region of the cDNA seems to be one of the longest among the cDNAs heretofore isolated. A comparison of the brain cDNA sequence (2424 bp) with the corresponding region of the liver cDNA (1574 bp) revealed differences in 5 nucleotides. The brain clone contained A----G and C----G base differences from the hepatic sequence, resulting in amino acid changes from Tyr and Arg in the liver clone, to Cys and Gly in the brain clone, respectively. The other 3 nucleotide differences are silent with respect to the amino acid sequence of the protein. Therefore, the amino acid sequence of the brain argininosuccinate lyase, as deduced from the nucleotide sequence of its cDNA clone, was identical with that of the liver protein, except for two amino acid residues. These minor changes may reflect a microheterogeneity of the argininosuccinate lyase gene. The brain and liver enzymes seem to be encoded by the same structural gene.
Assuntos
Argininossuccinato Liase/genética , Encéfalo/enzimologia , DNA/análise , Fígado/enzimologia , Liases/genética , Sequência de Aminoácidos , Animais , Argininossuccinato Liase/análise , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , RatosRESUMO
Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.
Assuntos
Acetil-CoA C-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Mitocôndrias/enzimologia , Ornitina Carbamoiltransferase/metabolismo , Zoogloea/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico/fisiologia , Células Cultivadas , DNA/genética , Microcorpos/enzimologia , Plasmídeos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Zoogloea/genéticaRESUMO
A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene. The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined. The sequence showed no apparent similarity with that of the E. coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600. The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences. An mRNA of 2.0 kb was detected on Northern blotting analysis. The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K. et al. (1989) Nature 340, 478-483; Bernstein, H.D. et al. (1989) Nature 340, 482-486). We designated this gene as SRH1 (SRP54 homologue). Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.
Assuntos
Proteínas Fúngicas , Genes Fúngicos , Mamíferos/genética , Sinais Direcionadores de Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Partícula de Reconhecimento de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , DNA Fúngico/genética , Desoxirribonuclease HindIII/metabolismo , Retículo Endoplasmático/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Translocação GenéticaRESUMO
Argininosuccinate lyase [EC 4.3.2.1] is an enzyme of the urea cycle in the liver of ureotelic animals. The enzymes of the urea cycle, including argininosuccinate lyase, are regulated developmentally and in response to dietary and hormonal changes, in a coordinated manner. The nucleotide sequence of rat argininosuccinate lyase cDNA, which was isolated previously (Amaya, Y., Kawamoto, S., Oda, T., Kuzumi, T., Saheki, T., Kimula, S., & Mori, M. (1986) Biochem. Int. 13, 433-438), was determined. The cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,549), a 5'-untranslated sequence of 150 bp, and a 3'-untranslated sequence of 41 bp. The amino acid composition of rat liver argininosuccinate lyase predicted from the cDNA sequence is in close agreement with that determined on the purified enzyme. The predicted amino acid sequences of the human and yeast enzymes along the entire sequences (94 and 39%, respectively), except for a region of 66 residues of the human enzyme near the COOH terminus. However, the sequence of this region of the human enzyme predicted from another reading frame of the human enzyme cDNA is homologous with the corresponding sequences of the rat and yeast enzymes. Therefore, the human sequence should be re-examined. Lysine-51, the putative binding site for argininosuccinate, and the flanking sequences are highly conserved among the rat, steer, human, and yeast enzymes.
Assuntos
Argininossuccinato Liase/genética , DNA/genética , Fígado/enzimologia , Liases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Humanos , Dados de Sequência Molecular , Plasmídeos , Ratos , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluorescence studies.
Assuntos
Fosfatase Alcalina/isolamento & purificação , Glicosilfosfatidilinositóis/isolamento & purificação , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , Expressão Gênica , Glicosilfosfatidilinositóis/química , Humanos , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SpodopteraRESUMO
A protease that cleaves the precursor of ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, has been partially purified from the matrix fraction of rat liver mitochondria. The protease cleaved the precursors of several other matrix proteins at apparently correct sites. The protease was inhibited by 1,10-phenanthroline and EDTA, was reactivated by excess Mn2+ or Co2+, and did not cleave the alkali-denatured precursor proteins. These and other results indicate that this protease is responsible for the processing of at least several matrix protein precursors, and that the enzyme recognizes some three-dimensional conformation of the precursors as well as the amino acid sequences around the cleavage sites.
Assuntos
Mitocôndrias Hepáticas/enzimologia , Peptídeo Hidrolases/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Catálise , Quelantes/farmacologia , Cobalto/farmacologia , Reativadores Enzimáticos , Técnicas In Vitro , Manganês/farmacologia , Ornitina Carbamoiltransferase/metabolismo , Inibidores de Proteases , Desnaturação Proteica , RatosRESUMO
Ornithine carbamoyltransferase of rat liver mitochondrial matrix (subunit Mr = 36,000) is synthesized extra-mitochondrially as a larger precursor (subunit Mr = 39,400) which is transported into mitochondria, in association with post-translational proteolytic processing. Rat liver mitochondria convert the precursor to the mature enzyme as well as to a 37,000-Mr product, a possible intermediate of the processing [Mori, M., Miura, S., Tatibana, M., and Cohen, P.P. (1980) Proc. Natl Acad. Sci. USA, 77, 7044-7048]. A protease responsible for the conversion of the precursor to the 37,000-Mr product was purified 140-fold from the matrix fraction of rat liver mitochondria. The protease had an estimated Mr of 108,000 and an apparent pI of 5.5. Mature ornithine carbamoyltransferase (0.5 microgram) did not inhibit the cleavage of the precursor by the protease and presumably the latter cleaves a specific site on the extrapeptide of the carbamoyltransferase precursor. The protease was inhibited by metal-chelating reagents such as EDTA, o-phenanthroline and zincon and by a high concentration (1 mM) of leupeptin. It did not cleave several of the protein and peptide substrates tested including the precursor of mitochondrial carbamoyl-phosphate synthetase I. Apparently the same protease activity is widely distributed among mitochondria of rat kidney, spleen, heart and ascites tumor cells, all of which lack ornithine carbamoyltransferase. A possible physiological role of the protease in the processing of the mitochondrial protein precursors is discussed.
Assuntos
Precursores Enzimáticos/metabolismo , Mitocôndrias/enzimologia , Ornitina Carbamoiltransferase/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Animais , Fenômenos Químicos , Química , Cinética , Masculino , Camundongos , Peso Molecular , Peptídeo Hidrolases/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The functions and expression pattern of urea cycle enzymes have undergone considerable changes during the course of evolution. Sequence analyses shows that urea cycle enzymes from mammals are homologous to microbial enzymes of the arginine-metabolic pathway. Recently, an unexpected relationship was found between argininosuccinate lyase (EC 4.3.2.1), the fourth enzyme of the cycle, and delta-crystallin, a lens structural protein of birds and reptiles.
Assuntos
Evolução Biológica , Genes , Ureia/metabolismo , Animais , Arginase/genética , Argininossuccinato Liase/genética , Argininossuccinato Sintase/genética , Carbamoil-Fosfato Sintase (Amônia)/genética , Ornitina Carbamoiltransferase/genéticaRESUMO
cDNA clones for rat argininosuccinate lyase, a urea cycle enzyme, were cloned and amino acid sequence of the enzyme was predicted. The rat enzyme is 54% identical with the yeast enzyme, which is involved in arginine biosynthesis, thereby indicating that this urea cycle enzyme evolved from the arginine biosynthetic enzyme. A striking similarity (64% identity) was found between amino acid sequences of rat argininosuccinate lyase and chicken delta-crystallin, a major structural protein of the eye lens. The gene for the rat argininosuccinate lyase was cloned and its structure was determined. This gene is a single-copy gene about 14 kilobases long and is split into 16 exons. A comparison with chicken delta-crystallin genes revealed that all introns interrupt the protein-coding regions at homologous positions. This close similarity in structural organization provides strong evidence for the view that the chicken delta 1- and delta 2-crystallin genes evolved by recruitment and duplication of the preexisting argininosuccinate lyase gene and that delta 2-crystallin is probably the direct homologue of argininosuccinate lyase.
Assuntos
Argininossuccinato Liase/genética , Evolução Biológica , Cristalinas/genética , Genes , Liases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Humanos , Isoenzimas/genética , Dados de Sequência Molecular , Ratos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , SoftwareRESUMO
Messenger RNA for 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid beta-oxidation, was purified from livers of di(2-ethylhexyl)phthalate-treated rats by immunoadsorption of hepatic free polysomes to fixed cells of Staphylococcus aureus and enrichment for poly(A)-rich RNA by oligo(dT)-cellulose chromatography. Plasmid cDNA was constructed from this poly(A)-rich RNA by a modification of the method of Okayama and Berg and was transformed into the Escherichia coli DH1 strain. Plasmids containing cDNA sequences coding for 3-hydroxyacyl-CoA dehydrogenase were screened by differential colony hybridization, and were identified by hybrid-arrested translation and hybrid-selected translation. Plasmid pHADH-1, which contains a 1400-base-pair insert, hybridized to rat 3-hydroxyacyl-CoA dehydrogenase mRNA with a length of 1700 bases. Determination of the dehydrogenase mRNA by in vitro translation and dot-blot analysis with the cDNA probe showed that the induction of the enzyme in rat liver by di(2-ethylhexyl)phthalate could be attributed to an increase in the mRNA concentration.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Mitocôndrias Hepáticas/enzimologia , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Clonagem Molecular , DNA/isolamento & purificação , Enzimas de Restrição do DNA , DNA Recombinante , Dietilexilftalato/farmacologia , Imunoquímica , Técnicas In Vitro , Masculino , Hibridização de Ácido Nucleico , Plasmídeos , Poli A/análise , Biossíntese de Proteínas , RNA/análise , Ratos , Ratos EndogâmicosRESUMO
Using a baculovirus expression vector system, the alpha 1 subunit of the mouse AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-selective glutamate receptor channel was expressed in insect Spdoptera frugiperda cells. Binding studies using [3H]AMPA showed that insect cells infected with the recombinant virus expressed approximately 1.8 x 10(5) binding sites per cell on their surface. The ligand binding characteristics of the receptors expressed in insect cells were examined. The baculovirus-insect cell expression system affords high-efficiency expression of the receptor in sufficient amounts to permit structural and functional analyses.
Assuntos
Baculoviridae/genética , Ácido Ibotênico/análogos & derivados , Canais Iônicos/metabolismo , Receptores de Neurotransmissores/genética , Animais , DNA Recombinante , Expressão Gênica , Vetores Genéticos , Glutamatos/metabolismo , Glutamatos/farmacologia , Ácido Glutâmico , Ácido Ibotênico/metabolismo , Ácido Ibotênico/farmacologia , Insetos/genética , Insetos/metabolismo , Ácido Caínico/metabolismo , Ácido Caínico/farmacologia , Cinética , Camundongos , Ácido Quisquálico/farmacologia , Receptores de Glutamato , Receptores de Neurotransmissores/efeitos dos fármacos , Receptores de Neurotransmissores/metabolismo , Transfecção , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
The synthesis and intracellular transport of the mitochondrial matrix enzymes ornithine transcarbamylase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) and carbamoyl-phosphate synthetase (ammonia) I [carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] were studied in isolated rat hepatocytes. In pulse experiments at 37 degrees C, the larger precursors of the two enzymes appeared in the cytosol of the liver cells, where radioactivity levels of the precursors reached a plateau in 10-20 min after the pulse. The pulse-labeled mature enzymes appeared in the particulate fraction (containing mitochondria) after a time lag and increased almost linearly with time up to 40 min. The specific radioactivities of the precursors in the cytosol were much higher than those of the mature enzymes in the particulate fraction. In pulse--chase experiments, the labeled precursors disappeared from the cytosol with estimated half-lives of about 1-2 min. These results indicate that ornithine transcarbamylase and carbamoyl-phosphate synthetase I are initially synthesized as larger precursors and exist in a cytosolic pool from which they are transported into mitochondria and processed there to the mature enzymes concomitantly with or immediately after transport. Although the rates of synthesis, transport, and processing were decreased about 3-fold at 25 degrees C (as compared to incubation at 37 degrees C), the pool size of the precursors in the cytosol were somewhat larger at this temperature.