RESUMO
We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/química , Receptores de AMPA/metabolismo , Medula Espinal/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte/química , Proteínas de Transporte/genética , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Sinapses/ultraestrutura , Transcrição GênicaRESUMO
In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.
Assuntos
Trifosfato de Adenosina/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Receptores de AMPA/fisiologia , Proteínas de Transporte Vesicular , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Precipitação Química , Dendritos/metabolismo , Interações Medicamentosas , Proteínas Sensíveis a N-Etilmaleimida , Neurônios/metabolismo , Proteínas Qa-SNARE , Ratos , Ratos Sprague-Dawley , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Leveduras/genéticaRESUMO
The uptake of l-[(35)S]cystine was studied in six cystinotic and six normal fibroblast lines grown for five days either on cover slips or in 32-oz plastic flasks. Cystinotics showed greater uptake than normals. The apparent K(t) for cystine entry in both types of cells was 0.043 mM but cystinotic cells showed a higher maximum velocity of entry. A comparison of the fate of l-[(35)S]cystine incubated for 20 min with monolayers of cells showed 30% and 15% of the intracellular (35)S to be l-cystine in cystinotic and normal cells, respectively. The (35)S effluxed more slowly from cystinotic than from normal cells after a 20-min preloading with l-[(35)S]cystine. Identification of (35)S compounds in efflux media after 3 min showed 75% of the total (35)S was l-cystine with the remainder in cysteine and acidic sulfur metabolites of cystine with no essential difference between cystinotics and normals. In paired experiments, the specific activity of the effluxed l-[(35)S]cystine after both efflux periods was the same as that entering the cell, thus indicating that the free l-[(35)S]cystine had not exchanged with the pre-existing pool in the cystinotic cells. During 3 min efflux, the l-cystine pool in normal cells was depleted mainly by loss of free cystine. In cystinotic cells, a new steady state was attained after 21 min of efflux and the intracellular l-[(35)S]cystine had the same percentage of total radioactivity seen after the initial 20-min uptake. After the rapid efflux of l-[(35)S]cystine from normals, [(35)S]cysteine and other labeled cystine metabolites appeared in the efflux media. By the end of a 3-min efflux, cystinotic cells had incorporated more label into reduced glutathione than had normal cells. However, when the new steady state was attained in cystinotics, the amounts of (35)S in glutathione were not markedly different in the two types of cells. Approximately 95% of the total label could be accounted for in free sulfur compounds. The data show an increased uptake and decreased efflux of cystine from cystinotic cells. However, it is not possible to conclude if these differences are due to primary changes in membrane function or to the reflection of metabolic defects without further investigation.
Assuntos
Cistina/metabolismo , Cistinose/metabolismo , Fibroblastos/metabolismo , Adolescente , Transporte Biológico , Radioisótopos de Carbono , Células Cultivadas , Criança , Pré-Escolar , Meios de Cultura , Cisteína/metabolismo , Glutationa/metabolismo , Humanos , Técnicas In Vitro , Lactente , Cinética , Metionina/metabolismo , Radioisótopos de Enxofre , Fatores de Tempo , TrítioRESUMO
DNA nick end-labeling (TUNEL) and heat shock protein (HSP)70 immunocytochemistry were performed on the same brain sections 1 (n = 6), 3 (n = 12), and 7 (n = 7) days following permanent middle cerebral artery (MCA) occlusions produced in adult rats using the endovascular carotid suture method. In the cortex at 1 and 3 days following MCA occlusions, HSP70 immunoreactive neurons were located outside areas of infarction and showed little evidence of DNA fragmentation. HSP70-stained cortical neurons were intermingled with TUNEL cells near the infarct, but extended for greater distances away from the infarct. DNA fragmentation occurred in CA1 hippocampal neurons in 39% of the animals at 1 and 3 days following ipsilateral MCA occlusion. Bilateral DNA fragmentation occurred in CA1 neurons in one subject. HSP70 protein was expressed in CA1 hippocampal neurons in nine of 18 (50%) animals at 1 and 3 days following MCA occlusions, including all animals that exhibited hippocampal DNA fragmentation. Three animals had bilateral expression of HSP70 in CA1 neurons. Cells that stained for either HSP70 protein or DNA fragmentation existed in close proximity to one another. Approximately 5-7% of HSP70-stained cells were TUNEL stained and 3% of TUNEL-positive cells also stained for HSP70. There was no HSP70 staining or DNA fragmentation in the brains of sham-operated controls (n = 4) or in the brains of animals 7 days following MCA occlusions. These data suggest that ischemic cells capable of translating HSP70 protein generally do not undergo DNA fragmentation. These data support the concept that most HSP70 protein-containing neurons in the cortical "penumbra" and hippocampus survive ischemic injury and are "reversibly injured." It is shown that CA1 hippocampal pyramidal neurons die or are reversibly injured in approximately 50% of animals following permanent MCA occlusions. Although the mechanism of this hippocampal injury is unknown, it could relate to transynaptic activation of N-methyl-D-aspartate (NMDA) receptors that mediate induction of early genes in hippocampus.
Assuntos
Isquemia Encefálica/patologia , Artérias Cerebrais/patologia , Córtex Cerebral/metabolismo , Dano ao DNA , Proteínas de Choque Térmico HSP70/biossíntese , Hipocampo/metabolismo , Animais , Apoptose/genética , Isquemia Encefálica/metabolismo , Córtex Cerebral/patologia , Hipocampo/patologia , Masculino , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-DawleyRESUMO
The prolonged expression of the leucine zipper fos/jun immediate early genes (IEG) has been correlated with neuronal death after cerebral ischemia. In this study, the expression of six zinc finger IEG was examined using in situ hybridization in adult rats after middle cerebral artery occlusion (MCAO) with the suture model. NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3, and Nurr1 mRNA were all induced throughout the ipsilateral cortex at 1 hour to 12 hours after MCAO. The cortical induction for most of the genes was greatest in the anterior cingulate and the anterior cerebral artery (ACA) and middle cerebral artery (MCA) transition zone. All of the zinc finger IEG were induced at 1 hour in all regions of hippocampus. NGFI-A and NGFI-B were induced in ipsilateral thalamus. Within areas of infarction, the basal IEG mRNA expression, and expression of the housekeeping gene cyclophilin A mRNA, decreased below control levels by 12 hours after the ischemia. Immediate early gene expression outside areas of infarction returned to control levels in most brain regions by 24 hours except for egr-3, which continued to be induced in the MCA/ ACA transition zone for 24 hours, and NGFI-A, which continued to be expressed in specific regions of the thalamus for 72 hours. The induction of these IEG in the cortex is likely caused by ischemia-induced cortical spreading depression, with the hippocampal and thalamic IEG induction being caused by activation of efferent cortical pathways to these regions. The prominent induction of NGFI-B, NGFI-C, egr-2, and egr-3 in the anterior cingulate cortex, the ACA/MCA transition zone, and medial striatum could reflect the ischemic regions around MCA infarcts. The prolonged NGFI-A expression observed in thalamus in this study, and in CA1 of hippocampus after global ischemia in the gerbil in a previous study, suggests that the prolonged NGFI-A, expression could be the result of or the cause of the delayed cell death. Prolonged NGFI-A expression, like c-fos and c-jun, seems to provide a marker for slowly dying neurons.
Assuntos
Isquemia Encefálica/genética , Proteínas Imediatamente Precoces/análise , Dedos de Zinco/genética , Isomerases de Aminoácido/genética , Animais , Proteínas de Transporte/genética , Núcleo Caudado/química , Córtex Cerebral/química , Proteínas de Ligação a DNA/análise , Proteína 1 de Resposta de Crescimento Precoce , Proteína 2 de Resposta de Crescimento Precoce , Endodesoxirribonucleases/genética , Expressão Gênica , Genes fos/genética , Genes jun/genética , Hipocampo/química , Hibridização In Situ , Dados de Sequência Molecular , Peptidilprolil Isomerase , Putamen/química , RNA Mensageiro/análise , Ratos , Tálamo/química , Fatores de Transcrição/análise , Córtex Visual/químicaRESUMO
Cystinotic fibroblasts when incubated in complete medium with 100 microM chloroquine rapidly lose their stored cystine, 50% or more disappearing in 2 hr and 80% in 4 hr. During the same 2 to 4 hr period of incubation with chloroquine, no change in intracellular pools of acidic and neutral amino acids was observed. However, after 5 hr the cellular pools of these amino acids became diminished and by 24 hr became markedly depleted. Chloroquine inhibited the uptake of 35S cystine by cystinotic cells during one hour incubations in phosphate buffered saline medium. Chloroquine altered the intracellular fate of exogenous 35S cystine into cystine, cysteine and glutathione (GSH) in cystinotic cells grown in both complete medium for 24 hr or placed in buffered saline for 2 hr. In the complete medium, chloroquine suppressed incorporation of label into cystine, cysteine and GSH pools after 1 hr. In the short-term study, the drug prevented the incorporation of label into cystine and cysteine pools after 45 min.
Assuntos
Cloroquina/farmacologia , Cistina/metabolismo , Cistinose/metabolismo , Aminoácidos/metabolismo , Criança , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , HumanosRESUMO
The transport of the amino acids, cystine and lysine, was studied in epithelial cell lines propagated from human kidney cortex. Cystine uptake data were reproducible in different cell lines and did not vary over several cell passages of an individual cell line. The transport of this disulfide amino acid was sodium-dependent with kinetic analysis showing one apparent Kt system of 0.09 mmol/L and Vmax of 0.054 mmol/L cell water/min. Studies of the kinetics of lysine transport, however, revealed two uptake systems with apparent high and low affinities with Kt of 0.14 mmol/L and 5 mmol/L and Vmax of 0.041 and 0.167 mmol/L cell water/min, respectively. Glutamate appeared to be the most potent inhibitor of cystine uptake by these cultured human renal cells and this interaction was competitive. Although cystine did not inhibit lysine uptake, arginine and ornithine were shown to be major inhibitors, thus providing evidence for the presence of a shared dibasic amino acid transport system.
Assuntos
Cistina/metabolismo , Rim/metabolismo , Lisina/metabolismo , Arginina/metabolismo , Ligação Competitiva , Transporte Biológico , Epitélio/metabolismo , Humanos , Rim/citologia , Cinética , Ornitina/metabolismo , Sódio/metabolismoRESUMO
The effect of cystine dimethylester on the renal handling of phosphate, glucose, alpha-amino nitrogen, amino acids, and protein in vivo and on the uptake of lysine, glycine, taurine, and alpha-methyl glucoside by isolated renal tubules in vitro was studied in adult male rats. Parenteral administration of 400 mumol twice a day for four days of cystine dimethylester led to an increased urine volume, and excretion of phosphate, glucose, alpha-amino nitrogen, and the amino acids glutamine, proline, alanine, 1/2 cystine, ornithine, lysine, histidine, and glycine. Cystine dimethylester treatment did not affect the creatine clearance nor were any renal anatomic abnormalities noted. Intracellular cysteine, but not cystine, was increased in the kidney after the four days of treatment. Pre-incubation of isolated renal tubules with 2 mmol/L cystine dimethylester for ten minutes markedly inhibited the uptake of 0.025 mmol/L lysine, 0.1 mmol/L glycine, 0.01 mmol/L taurine, and 2 mmol/L alpha-methyl glucoside. Incubation with 2 mmol/L cystine dimethylester for ten minutes did not affect the ability of the renal tubule to exclude trypan blue dye, although longer incubation times did lead to significant staining. The intracellular cystine concentration of the renal tubule did rise significantly after incubation with cystine dimethylester, a biochemical correlate of the human disease cystinosis. These studies indicate that cystine dimethylester can induce an experimental form of the Fanconi syndrome both in vivo and in vitro and offers a new model for investigating the mechanisms underlying this enigmatic disorder.
Assuntos
Cistina/análogos & derivados , Modelos Animais de Doenças , Síndrome de Fanconi/metabolismo , Túbulos Renais Proximais/metabolismo , Aminoácidos/metabolismo , Animais , Cistina/farmacologia , Cistina/toxicidade , Cistinose/induzido quimicamente , Cistinose/metabolismo , Síndrome de Fanconi/induzido quimicamente , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Metilglucosídeos/metabolismo , Ratos , Ratos EndogâmicosRESUMO
In previous work it has been shown that adult male, but not female, offspring of rats that have been injected with protamine zinc insulin (6 IU/kg) on days 15-20 of gestation, develop significant obesity beginning about 50 days of age. This obesity is accompanied by elevated medial hypothalamic extracellular norepinephrine levels. To examine whether the expression of obesity in male offspring is mediated by perinatal testosterone levels, male offspring of insulin-treated or control dams were either castrated or received sham surgery on postnatal day 1. Castrated male offspring of insulin-treated dams did not become obese like their gonadally intact male littermates. This suggests that perinatal testosterone levels may interact with developmental processes mediating the obesity in male offspring of insulin-treated dams. A second question addressed was whether the elevated hypothalamic extracellular norepinephrine levels observed in our earlier work are evident as morphological changes in norepinephrine-containing systems in the medial hypothalamus and locus coeruleus. We found a significant enhancement of dopamine-beta-hydroxylase immunoreactivity in fibers innervating the paraventricular nucleus of the hypothalamus in 121-day-old, gonadally intact male offspring of insulin-injected dams. This suggests that the impact of maternal insulin injections on offspring obesity may be mediated through its organizing action on feeding-related fibers in the paraventricular nucleus.
Assuntos
Peso Corporal/fisiologia , Hipotálamo/embriologia , Insulina/sangue , Norepinefrina/fisiologia , Efeitos Tardios da Exposição Pré-Natal , Animais , Dopamina beta-Hidroxilase/análise , Comportamento Alimentar/fisiologia , Feminino , Gônadas/fisiologia , Gônadas/cirurgia , Hipotálamo/química , Hipotálamo/enzimologia , Imuno-Histoquímica , Masculino , Vias Neurais , Obesidade/embriologia , Obesidade/fisiopatologia , Orquiectomia , Gravidez , Prosencéfalo/química , Prosencéfalo/embriologia , Prosencéfalo/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
A study of the lysosomal hydrolases bete-galactosidase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and arylsulphatases A and B has been carried out on fibroblasts cultured from seven patients with cystinosis and eight control subjects. beta-Galactosidase activity was found to be consistently lower in cells derived from cystinotics, while the other enzymes studied showed no significant differences between normals and cystinotics.
Assuntos
Cistinose/enzimologia , Hidrolases/metabolismo , Arilsulfatases/metabolismo , Células Cultivadas , Criança , Cistina/metabolismo , Fibroblastos/enzimologia , Galactosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , História do Século XVIII , Humanos , Lisossomos/enzimologia , Mitocôndrias/enzimologiaRESUMO
Sprague-Dawley rats anesthetized with isoflurane, underwent MCA occlusion by intraluminal 3-0 suture insertion, either 22 mm (n = 8) or 18 mm (n = 6) beyond the CCA bifurcation or were sham-operated as controls (n = 3) for autoradiographic analysis of cerebral blood flow. Infarct volume was measured 24 hours after the onset of ischemia (22 mm, n = 11; 18 mm, n = 10); neurological examinations were performed at 6 and 24 hours. Cerebral blood flow in the MCA distribution was significantly lower in the 22 mm suture insertion group than in the 18 mm group (p < 0.05). The total infarct volume was significantly larger (197 +/- 15 versus 135 +/- 19 mm3, p < 0.05) and the coefficient of variance was significantly smaller (23.8% versus 43.9%, p < 0.05) in the 22 mm group. Border zone regions of medial caudoputamen and dorsolateral cortex were often spared in the 18 mm group but never in the 22 mm group. The neurological deficit was more severe in the 22 mm group at 24 hours (p < 0.05), but not at 6 hours. The greater blood flow reduction and the less variable histological damage in dorsolateral cortex (a watershed area between the middle and anterior cerebral arteries) and the greater histological damage in medial caudate in the 22 mm group further characterizes this focal ischemia model for two potential applications: 22 mm insertion for studies requiring extensive and reproducible infarcts; 18 mm insertion for studies requiring less severe and more variable lesions after permanent MCA occlusion.
Assuntos
Arteriopatias Oclusivas/fisiopatologia , Isquemia Encefálica/fisiopatologia , Artérias Cerebrais/cirurgia , Infarto Cerebral/fisiopatologia , Circulação Cerebrovascular/fisiologia , Animais , Arteriopatias Oclusivas/complicações , Isquemia Encefálica/complicações , Infarto Cerebral/etiologia , Masculino , Ratos , Ratos Sprague-Dawley , SuturasAssuntos
Alanina/análise , Cisteína/análise , Cistina/análise , Fibroblastos/análise , Ácidos Sulfínicos/análise , Taurina/análise , Cromatografia por Troca Iônica , Cromatografia em Papel , Cistinose/metabolismo , Humanos , Métodos , Pele , Espectrofotometria , Ácidos Sulfônicos/análise , Isótopos de EnxofreRESUMO
The metabolic fate of 15N-labeled glutamine and glutamate in cultured human renal cortical epithelial cells was investigated. The main goal was to elucidate the major pathways of ammoniagenesis depending on varying H+ concentration. Incubations at pH 7.4 or 6.8 were conducted with either 1 mM [5-15N]glutamine, [2-15N]glutamine, [15N]glutamate, or L-[2-15N]-gamma-glutamylmethylamide. The results demonstrate that acute acidosis had little effect on total ammonia generation from glutamine. However, 15NH3 formation from [5-15N]glutamine was significantly higher at pH 7.4 compared with pH 6.8. Conversely, at pH 6.8, 15NH3 production from either [2-15N]-glutamine or [15N]glutamate was twofold higher than at pH 7.4. Thus the observations indicate that acute acidosis had little effect on net ammonia production from glutamine due to decreased flux through glutaminase and concomitant increased flux through glutamate dehydrogenase. When L-[2-15N]-gamma-glutamylmethylamide was utilized as the sole substrate, significantly higher amounts of 15NH3 and 15N-labeled amino acids were formed at pH 6.8 compared with pH 7.4. Addition of either 1 mM pyruvate or alpha-ketoglutarate significantly decreased 15NH3 and increased 15N-amino acid formation from either [2-15N]glutamine or [2-15N]-gamma-glutamylmethylamide. The metabolism of either substrate via transamination reaction was significantly stimulated at acidic pH, presumably due to a depleted pool of alpha-ketoglutarate during the course of the incubations. The data indicate that in addition to glutaminase I and glutamate dehydrogenase, the glutamine aminotransferase (glutaminase II) pathway exists in cultured human renal cells. The data suggest that glutamate dehydrogenase flux and/or the alpha-ketoglutarate dehydrogenase reaction may have an important regulatory role in ammoniagenesis from glutamine and/or glutamate in human kidney during acute acidosis.
Assuntos
Amônia/biossíntese , Córtex Renal/citologia , Células Cultivadas , Epitélio/metabolismo , Glutamatos/metabolismo , Ácido Glutâmico , Glutamina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Isótopos de Nitrogênio , Piruvatos/metabolismo , Ácido PirúvicoRESUMO
The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.
Assuntos
Aminoácidos/farmacocinética , Cistina/farmacocinética , Rim/citologia , Gambás/metabolismo , Aminoácidos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cistina/metabolismo , Relação Dose-Resposta a Droga , Rim/metabolismo , Sódio/farmacologia , Radioisótopos de EnxofreRESUMO
The characteristics of a fluorimetric method for assay of gamma-glutamyltranspeptidase (EC 2.3.2.2) in cultured human skin fibroblasts from normal donors and cystinotic patients using gamma-glutamyl-7-amino-4-coumarin as substrate are described. It is possible with this method to measure enzymatic activity in sonicates of cells with as little as 0.029 mmol/l L-cystine as acceptor-substrate with only 0.20 mg of cellular protein after 30--60 min of incubation. The properties of the enzyme in cells from patients with cystine storage disease show elevated gamma-glutamyltranspeptidase under various incubation conditions.
Assuntos
Cistinose/enzimologia , Pele/enzimologia , gama-Glutamiltransferase/metabolismo , Células Cultivadas , Criança , Cumarínicos/metabolismo , Cistina/metabolismo , Fibroblastos/enzimologia , Glicilglicina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , CinéticaRESUMO
1. Glutathione reductase and glutathione-cystine transhydrogenase activity in supernatant fractions of whole homogenates and homogenates of mucosal and muscular layers were determined in developing rat intestine after determination of the optimum conditions for assay of the two enzymes. In jejunum from adult rat, the K(m) values for GSSG reductase and GSH-cystine transhydrogenase activities were 0.25mm-GSSG and 0.23mm-cystine respectively. 2. The two activities could be differentiated by stability studies since GSSG reductase was stable at 60 degrees C for 10min and could be stored at 4 degrees C for 24h without loss of activity. GSH-cystine transhydrogenase, on the other hand, was denatured at 60 degrees C and completely inactive after 24h storage at 4 degrees C. 3. Based on calculations of total activities, both enzymes increased from the eighteenth day until the animals were young adults. 4. Total GSSG reductase activity increased at a greater rate with age than total GSH-cystine transhydrogenase activity as evidenced by activity ratios for GSH-cystine transhydrogenase/GSSG reductase of 0.44 and 0.12 in ileum from suckling and adult rats respectively, and 0.31 and 0.24 in jejunum from suckling and adult rats respectively. 5. In mucosa from adult rats GSSG reductase was more active in the ileum than in the jejunum, whereas GSH-cystine transhydrogenase activity was higher in the jejunum. 6. GSH-cystine transhydrogenase was active only in the muscle cells of the ileum of 7-day-old rats but became localized primarily in the mucosal layer in the adult rat. However, GSSG reductase activity was distributed evenly between the two layers throughout the intestine.
Assuntos
Glutationa Redutase/metabolismo , Intestino Delgado/enzimologia , Oxirredutases/metabolismo , Envelhecimento , Animais , Cistina , Feminino , Glutationa , Íleo/enzimologia , Íleo/crescimento & desenvolvimento , Mucosa Intestinal/enzimologia , Mucosa Intestinal/crescimento & desenvolvimento , Intestino Delgado/crescimento & desenvolvimento , Jejuno/enzimologia , Jejuno/crescimento & desenvolvimento , Cinética , Ratos , Espectrofotometria UltravioletaRESUMO
Cultured opossum kidney (OK) and porcine kidney (LLC-PK1) cells were compared for biochemical characteristics and cystine transport systems. The cell lines differ in amount of protein per cell, with OK cells having approximately one-half the amount found in LLC-PK1. Both cell lines contain 19 micrograms DNA/10(6) cells. As cells reach confluence, cystine uptake increases in OK and decreases in LLC-PK1 cells. Throughout the growth period, only lysine inhibits cystine uptake in OK, whereas glutamate is the inhibitor in LLC-PK1. The predominant site of cystine transport in OK cells is across the apical membrane, and the basolateral membrane is the corresponding site of transport in LLC-PK1 cells. Although the intracellular reduced glutathione pool is the same, the cysteine pool in OK cells is approximately one-fourth that found in LLC-PK1 cells. The ability of OK cells to reflect the shared cystine-dibasic amino acid transport system and LLC-PK1 to exhibit the cystine-glutamate antiporter system makes available two models for investigation of the development and structure of cystine transport systems.