RESUMO
Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline.
Assuntos
Glutationa Sintase/deficiência , Peptídeo Sintases/deficiência , Eritrócitos/enzimologia , Fibroblastos/enzimologia , Glutationa Sintase/genética , Humanos , Técnicas In Vitro , Leucócitos/enzimologia , Ácido Pirrolidonocarboxílico/sangue , Ácido Pirrolidonocarboxílico/urinaRESUMO
Two isoforms of divalent metal transporter 1 (DMT1) (Nramp2 and DCT1) are encoded by two mRNA species, one of which contains an iron response element (IRE) motif in the 3'-noncoding region. The subcellular distribution of the two isoforms of DMT1 is distinct, and the -IRE species accumulates in the nucleus of neuronal or neuronal-like cells. Reverse transcription-PCR and Western blot analysis of PC12 cells reveals that these cells express both forms of DMT1. Immunofluorescence and immunoblotting studies, using immunospecific antibodies to the -IRE form of DMT1, demonstrate that this form of the transporter, in PC12 cells, is predominantly localized in the nucleus, cell membrane, and neurites with only weak staining of the cell body. Studies using antibodies to the +IRE form indicate that this species of DMT1 is distributed within vesicles in the cell body and neurite projections, with minimal nuclear staining. Similar staining patterns are observed for the two forms of DMT1 in cultures of sympathetic ganglion neurons isolated from perinatal rat pups. To determine whether nuclear localization of the -IRE form of DMT1 is constrained to neuronal or neuronal-like cells, immunocytochemical studies were performed with human embryonic kidney 293T (HEK293T), HEP2G hepatoma and medulloblastoma, and rat Schwann cells. The -IRE-specific antibodies stained nuclei from medulloblastoma, whereas little nuclear staining was observed with HEK293T, hepatoma, or Schwann cells. The unexpected finding that the -IRE species of DMT1 selectively accumulates in the nucleus of neuronal and neuronal-like cells leads us to postulate that the two proteins may have different functions in vivo.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Proteínas de Ligação ao Ferro , Proteínas de Membrana/metabolismo , Células PC12/metabolismo , Sistema Nervoso Simpático/metabolismo , Animais , Sítios de Ligação/genética , Transporte Biológico , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Imunofluorescência , Humanos , Immunoblotting , Imuno-Histoquímica , Ferro/metabolismo , Proteínas de Membrana/genética , Neuritos/metabolismo , Células PC12/citologia , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores da Transferrina/metabolismo , Sistema Nervoso Simpático/citologiaRESUMO
The Belgrade rat has a microcytic, hypochromic anemia inherited as an autosomal recessive trait (gene symbol b). Transferrin-dependent iron uptake is defective because of a mutation in Nramp2 (now DMT1, also called DCT1), the protein responsible for endosomal iron efflux. Hence, Belgrade reticulocytes are iron deficient. We show that a chromatographic method is able to measure the amount of 'free' heme in reticulocytes. Most of the 'free' heme is the result of biosynthesis. Succinylacetone, an inhibitor of heme synthesis, decreases the level of 'free' heme and cycloheximide, an inhibitor of globin synthesis, increases the 'free' heme level. In a pulse-chase experiment with 59Fe-transferrin, the 'free' heme pool behaves as an intermediate, with a half-life of just over 2 h. Belgrade reticulocytes contain about 40% as much 'free' heme as do heterozygous or homozygous reticulocytes. This deficiency of 'free' heme slows initiation of translation in Belgrade reticulocytes by increasing the level of an inhibitor of initiation. Thus the Belgrade rat makes a whole animal model available with chronic heme deficiency.
Assuntos
Anemia Hipocrômica/genética , Heme/deficiência , Reticulócitos/metabolismo , Anemia Hipocrômica/sangue , Animais , Sistema Livre de Células , Cicloeximida/farmacologia , Modelos Animais de Doenças , Heme/biossíntese , Heptanoatos/farmacologia , Ferro/metabolismo , Ratos , Reticulócitos/efeitos dos fármacosRESUMO
The beta chain of rabbit (Oryctolagus caniculus) hemoglobin has previously been reported to contain a single residue of isoleucine at beta(112). We have detected other rabbits with either zero isoleucyl residues or half a residue per beta chain. This character is polymorphic and inherited as a simple mendelian autosomal codominant.-Normally the modal number of ribosomes per polyribosome is 4 to 6 in reticulocyte lysates; but incubation of rabbit reticulocytes prior to lysis with L-o-methylthreonine (OMT), an isostere of isoleucine, leads to a bimodal distribution in lysates with 2-3 and 8-12 ribosomes as modes. This alteration has been attributed to ribosomal traffic jams caused by starvation for ile-tRNA at mRNA codons corresponding to the locations of isoleucyl residues at positions alpha(10), alpha(17), alpha(55) and beta(112). We have confirmed this interpretation by incubating OMT with reticulocytes from rabbits with integral, half integral and nil values for isoleucyl residues per beta chain to show that formation of the larger clusters of polyribosomes requires that beta(112) = ile.
Assuntos
Hemoglobinas/biossíntese , Polimorfismo Genético , Polirribossomos/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Radioisótopos de Carbono , Centrifugação com Gradiente de Concentração , Genes Dominantes , Genótipo , Isoleucina/metabolismo , Fenótipo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Coelhos , Reticulócitos/citologia , Reticulócitos/metabolismo , Espectrofotometria Ultravioleta , Treonina , Trítio , ValinaRESUMO
Homozygous beta-thalassemic mice show many of the features seen in human beta-thalassemia, such as decreased hemoglobin, hematocrit, and red blood cell count as well as increased reticulocyte count. They also exhibit splenomegaly and a decrease in osmotic fragility of red cells. beta-thalassemic mice were examined for spontaneous iron overload at ages ranging from 20 to 595 days. Accumulation of iron was shown to occur in the spleen, liver, and kidneys but not in the heart. Sections of spleen, liver, kidney, and heart were stained for iron and subjectively scored. Image analysis microscopy was used to examine sections of spleen and liver. Nonheme iron in the four tissues was quantitated using the bathophenanthroline sulfonate colorimetric assay. An increase in tissue iron occurred primarily in the spleen, even before weaning, despite the low iron content of milk. Iron accumulation in the absence of blood transfusion is of interest because iron overload is the major cause of death in human beta-thalassemia.
Assuntos
Ferro/farmacocinética , Talassemia/metabolismo , Envelhecimento/metabolismo , Animais , Peso Corporal , Rim/metabolismo , Fígado/metabolismo , Camundongos , Microscopia/métodos , Tamanho do Órgão , Baço/metabolismoRESUMO
In this study, we investigated the cellular distribution of iron in the brain of Belgrade rats. These rats have a mutation in Divalent Metal Transporter 1, which has been implicated in iron transport from endosomes. The Belgrade rats have iron-positive pyramidal neurons, but these are fewer in number and less intensely stained than in controls. In the white matter, iron is normally present in patches of intensely iron-stained oligodendrocytes and myelin, but there is dramatically less iron staining in the Belgrade rat. Those oligodendrocytes that stained for iron did so strongly and were associated with blood vessels. Astrocytic iron staining was seen in the cerebral cortex for both normal rats and Belgrade rats, but the iron-stained astrocytes were less numerous in the mutants. Iron staining in tanycytes, modified astrocytes coursing from the third ventricle to the hypothalamus, was not affected in the Belgrade rat, but was affected by diet. The results of this study indicate that Divalent Metal Transporter 1 is important to iron transport in the brain. Iron is essential in the brain for basic metabolic processes such as heme formation, neurotransmitter production and ATP synthesis. Excess brain iron is associated with a number of common neurodegenerative diseases. Consequently, elucidating the mechanisms of brain iron delivery is critical for understanding the role of iron in pathological conditions.
Assuntos
Anemia Hipocrômica/metabolismo , Química Encefálica , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Proteínas de Ligação ao Ferro , Ferro/análise , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/deficiência , Ratos Mutantes/metabolismo , Substituição de Aminoácidos , Anemia Hipocrômica/genética , Animais , Astrócitos/química , Proteínas de Transporte/genética , Cruzamentos Genéticos , Dieta , Endossomos/metabolismo , Feminino , Masculino , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Oligodendroglia/química , Oligodendroglia/patologia , Especificidade de Órgãos , Mutação Puntual , Ratos , Ratos Endogâmicos F344 , Ratos WistarAssuntos
Hemoglobinas Anormais , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cromatografia Gasosa , Cromatografia em Gel , Cromatografia em Camada Fina , Formiatos , Variação Genética , Isoleucina , Lisina , Fragmentos de Peptídeos/análise , Espectrofotometria Ultravioleta , Tiocianatos , Trítio , ValinaAssuntos
Globinas/biossíntese , Hemoglobinas Anormais/biossíntese , Aminoácidos/análise , Animais , Feminino , Hemoglobina Fetal/biossíntese , Genótipo , Homozigoto , Humanos , Isoleucina/metabolismo , Lisina/metabolismo , Mutação , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Gravidez , Coelhos , Espectrofotometria Ultravioleta , Treonina/análogos & derivados , Treonina/farmacologia , Trítio , Valina/metabolismoAssuntos
Clorometilcetonas de Aminoácidos/farmacologia , Globinas/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos , Tosilina Clorometil Cetona/farmacologia , Animais , Cromatografia por Troca Iônica , Técnicas In Vitro , Metionina/análise , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismoRESUMO
Because small peptides are lost into the organic solvents used, it is virtually impossible to obtain the complete amino acid sequence of a small peptide using only an automated peptide sequencer of the spinning cup type. To overcome this problem we have extended peptides at the carboxy terminus by attachment to equine cytochrome c by a water soluble carbodiimide, relying on the acetylated N-terminus of the cytochrome to minimize its direct contribution to recovery of PTH-amino acids. The Model Peptide H-Leu-Trp-Met-Arg-phe-Ala-OH was used for most experiments. After reaction of 3H-peptide with cytochrome c, about one-third of the tritium counts migrated with cytochrome c during gel filtration. After attachment, the amino acid sequence of the hexapeptide was readily determined with a single cleavage Quadrol program in a Beckman 890B sequencer, whereas only the N-terminal residue was recovered without attachment. The repetitive yield after attachment was 95-96%, with 21-27+ overlap and an initial yield of 18-20%. Sequence data with other peptides illustrate applications and present limitations of our approach.
Assuntos
Sequência de Aminoácidos , Grupo dos Citocromos c , Peptídeos , Autoanálise/métodos , Cromatografia em Gel , Peptídeos/análise , SolventesRESUMO
With puromycin one can recognize when the synthesis of a given protein is dependent on amino acyl tRNA that is present in rate limiting amount. We demonstrate this use of puromycin by its interaction with another inhibitor, L-o-methylthreonine. L-o-methylthreonine lowers the Ile-tRNA concentration in the cell, thereby inhibiting synthesis of proteins containing isoleucine. In certain rabbits, the alpha hemoglobin chain has three isoleucyl residues and the beta chain none. L-o-methylthreonine thus inhibits alpha globin synthesis in intact reticulocytes from these rabbits. When puromycin and L-o-methylthreonine are used together, the two inhibitors synergize in inhibiting alpha globin synthesis. Hence, puromycin is a more effective inhibitor when the Ile-tRNA concentration is lowered. Cycloheximide and sodium fluoride have different modes of action from puromycin. Neither synergizes with L-o-methylthreonine; instead, the interaction is less than additive. We have found that beta chain synthesis in rabbit reticulocytes is more sensitive than alpha to inhibition by puromycin. This difference could reflect either differences in amino acid sequence or tRNA dependent limitations of beta chain elongation. The switch from fetal to adult hemoglobin in humans does not involve changes in limiting amino acyl tRNA because, for cord blood from infants of different developmental ages, the puromycin sensitivity of incorporation into gamma and beta chains remains constant.
Assuntos
Hemoglobina Fetal/biossíntese , Globinas/biossíntese , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Puromicina/farmacologia , Aminoacil-RNA de Transferência/metabolismo , Animais , Cicloeximida/farmacologia , Sinergismo Farmacológico , Fluoretos/farmacologia , Humanos , Isoleucina/metabolismo , Substâncias Macromoleculares , Coelhos , Treonina/análogos & derivados , Treonina/farmacologiaRESUMO
Tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits protein synthesis in intact cells and lysates. Its presence leads to polyribosome disaggregation. This inhibition is probably at the level of chain initiation because it does not slow poly(U)-dependent elongation of poly(F). This drug activates an inhibitor similar to the heme-controlled repressor in the postribosomal supernatant. High concentrations of ATP and GTP affect inhibitions of translation by the heme-controlled repressor and TLCK in a similar fashion. The latter also interferes with formation of the 40S initiation complex. Its action on translation can be prevented by preliminary incubation with thiols.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Proteínas Sanguíneas/biossíntese , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Reticulócitos/metabolismo , Tosilina Clorometil Cetona/farmacologia , Animais , Relação Dose-Resposta a Droga , Glutationa/farmacologia , Hemina/farmacologia , Cinética , Polirribossomos/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Coelhos , eIF-2 QuinaseRESUMO
Hemoglobin beta-chain synthesis by rabbit erythroid cells was tested for dependence on availability of complementary alpha-chains. Reticulocytes and bone marrow cells were obtained from variant rabbits that have hemoglobin with isoleucine in alpha-chains but not in beta-chains. This characteristic permits the use of L-O-methylthreonine, a specific isoleucine antagonist, to inhibit selectively the synthesis of hemoglobin alpha-chains without directly affecting that of beta-chains. Study of hemoglobin synthesis by bone marrow cells presents two problems that require careful management: (A) the fragility of the globin-synthesizing apparatus and (B) the isolation of globin from the various proteins made by the mixture of nucleated cells. Disruption of synthetic activity was minimized by collecting the bone marrow in autologous plasma then removing fat and connective tissue while the cells were suspended in this medium. Purification involved gel filtration of hemoglobin and globin then CM-cellulose chromatography of globin chains. Absence of radioactive isoleucine in beta-chains demonstrated the efficacy of this scheme in removing isoleucine-containing proteins that otherwise elute with beta-chains on CM-cellulose columns. In reticulocytes, when synthesis of alpha-chains is inhibited by 30%--80%, that of beta-chains is stimulated by 20%--60%, but in marrow cells, incorporation into beta-chains stays at control level when alpha incorporation is inhibited. The data indicate that beta-chain synthesis is independent of the availability of complementary alpha-chains.
Assuntos
Eritrócitos/análise , Hemoglobinas/biossíntese , Animais , Proteínas Sanguíneas , Células da Medula Óssea , Separação Celular , Globinas/isolamento & purificação , Hemoglobinas/metabolismo , Coelhos , Reticulócitos/análise , Treonina/análogos & derivados , Treonina/farmacologiaRESUMO
The amino acid sequences of the beta major and beta minor globin chains from DBA/2 mice have been determined. This information is of interest because DBA/2 mice are the strain of origin for most murine erythroleukemia lines. The primary structure of DBA/2 beta globins agrees completely with that predicted from the coding properties of BALB/c beta globin genes. This identity does not support a rapid evolutionary divergence in inbred mouse strains, at least at these loci in these strains.
Assuntos
Globinas/genética , Camundongos Endogâmicos DBA/genética , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Evolução Biológica , Camundongos , Camundongos Endogâmicos BALB C/genéticaRESUMO
We have examined the relationship between alpha and beta globin chain syntheses by utilizing the distribution of isoleucyl residues in rabbit hemoglobin. The alpha globin chain contains three isoleucyl residues while the beta chain of certain rabbits contains no isoleucine. O-Methyl-L-threonine, an isoleucine isostere, inhibits incorporation of radiolabeled amino acids into alpha chains in rabbit reticulocytes. When alpha chain synthesis is inhibited by 50-85%, beta synthesis is stimulated by 15-50%. The excess labeled beta chains are not distinguishable from authentic beta chains by any of the following criteria: (a) carboxymethyl cellulose chromatography in sodium phosphate-urea buffers, (b) electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, and (c) electrophoresis of methionine-containing tryptic peptides. The stimulation of beta synthesis continues after the pool of excess alpha chains has been exhausted by preincubation with O-methyl-L-threonine. The stimulation does not occur, however, when 1 mM 2-mercaptoethanol is added to the incubation medium or when the cells are excessively diluted in the incubation mixture. The rates of beta chain initiation and elongation during stimulation have been compared to the rates during normal synthesis. Although both rates are increased, the rate of elongation increases more than initiation, suggesting that initiation is the rate-limiting step in increased beta chain production. The stimulation of beta synthesis when alpha synthesis is inhibited is interpreted as resulting from relief of competition between alpha and beta mRNAs for limiting components of the protein synthetic apparatus.
Assuntos
Globinas/biossíntese , Aminoácidos/metabolismo , Animais , Heterozigoto , Homozigoto , Isoleucina/metabolismo , Lisina/metabolismo , Mercaptoetanol/farmacologia , Metionina/metabolismo , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fragmentos de Peptídeos/biossíntese , Polirribossomos/metabolismo , Coelhos , Reticulócitos/metabolismo , Treonina/análogos & derivados , Treonina/farmacologiaRESUMO
Belgrade rats have an autosomal recessive anemia with hypochromia and microcytosis. Iron uptake into reticulocytes is approximately 20% of normal, but transferrin uptake is unimpaired. We have systematically compared the transferrin cycle in Belgrade versus normal reticulocytes to locate the defect more precisely. Belgrade transferrin was functionally normal as purified transferrin or whole plasma. Transferrin affinity of Belgrade receptors was indistinguishable from normal, but Belgrade reticulocytes had twice as many receptors. Belgrade transferrin endocytosis was 1.5 times faster than normal, whereas exocytosis is about twice as fast. Initially Belgrade reticulocytes internalize iron at an unimpaired rate, but they lag behind normal by 5 min. During reincubation, they release 25-33% of iron taken up during a 30-min preincubation, whereas normal cells do not lose a detectable fraction. Unexpectedly, transferrin cycle time was unchanged. Hence another kinetic step of the cycle is slower, compensating for increases in Belgrade endocytosis and exocytosis. After one cycle, Belgrade reticulocytes retain only half of the iron that entered, but over 90% of iron entering normal cells remains within. Iron unloading is ineffective inside the Belgrade vesicle; 85% of iron that entered on transferrin returned to the medium after exocytosis, whereas only 45% of iron entering normal reticulocytes exits. Ineffective utilization of iron in or near Belgrade endosomes accounts for the Belgrade defect.
Assuntos
Reticulócitos/metabolismo , Transferrina/metabolismo , Anemia/genética , Anemia/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Endocitose , Exocitose , Ferro/metabolismo , Cinética , Ratos , Ratos MutantesRESUMO
Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: alpha, beta, and 2 gamma globin chains. We have verified that the latter two are the G gamma and A gamma globin chains which have respectively glycine or alanine at position 136. After incorporation of either [3H] alanine or [3H] glycine into newly synthesized globin each gamma chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the G gamma chain and alanine for the A gamma. Substantial metabolic conversion of [3H] glycine to serine and proline was also noted.