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During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.  相似文献   

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Cell-free extracts obtained from free-livingRhizobium sp. in early stationary phase had three times as much 5-aminolevulinate synthase activity as did similar extracts from log phase cells. The level of 5-aminolevulinate dehydratase was also elevated at this point. The presence of 0.1 mM hemin in the culture medium prevented the transitory increase in enzyme activities during this early stationary phase. The effect of hemin was counteracted by 1 mg bovine serum albumin per milliliter medium. This control of the development of 5-aminolevulinate synthase and 5-aminolevulinate dehydratase activities by free hemin suggests a mechanism by which heme and globin formation might be coordinated for the synthesis of leghemoglobin in legume root nodules.  相似文献   

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Succinylacetone was shown to inhibit aminolevulinate dehydratase (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24) to reduce cellular heme and porphyrins and to induce δ-aminolevulinate synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) in monolayers of chick embryo liver cells. Marked synergistic effects on δ-aminolevulinate synthase activity were obtained by combining succinylacetone with levulinate and porphyrogenic drugs. The time course of δ-aminolevulinate synthase activity showed a delayed synergistic response.  相似文献   

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Erythroid expression of the heme-regulated eIF-2 alpha kinase.   总被引:2,自引:0,他引:2       下载免费PDF全文
The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.  相似文献   

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Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.  相似文献   

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Coproporphyrinogen oxidase (EC 1.3.3.3.) catalyzes the sixth enzymic step of the heme biosynthetic pathway. Coproporphyrinogen oxidase activity is increased in mutant cells of Saccharomyces cerevisiae deficient in heme synthesis and this effect can be partially reversed by the addition of exogenous hemin. A similar increase is found in wild type yeast cells grown anaerobically. The strain-dependent increase varies between 5- and 40-fold. The activity changes are paralleled by similar changes in the steady-state amounts of coproporphyrinogen oxidase protein determined by immunoblotting and the steady-state concentrations of coproporphyrinogen oxidase mRNA estimated by in vitro translation/immunoprecipitation. This demonstrates that coproporphyrinogen oxidase synthesis is regulated by heme and oxygen at a pretranslational level in a negative fashion.  相似文献   

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Transcriptional control of rat heme oxygenase by heat shock   总被引:19,自引:0,他引:19  
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Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 microM hemin and 100 microM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 microM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40-60 microM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 microM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 microM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.  相似文献   

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The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS2 and CoCl2. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.  相似文献   

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5-Aminolevulinate synthase and the first step of heme biosynthesis   总被引:1,自引:0,他引:1  
5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate. In animals, fungi, and some bacteria, 5-aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway. Mutations on the human erythroid 5-aminolevulinate synthase, which is localized on the X-chromosome, have been associated with X-linked sideroblastic anemia. Recent biochemical and molecular biological developments provide important insights into the structure and function of this enzyme. In animals, two aminolevulinate synthase genes, one housekeeping and one erythroid-specific, have been identified. In addition, the isolation of 5-aminolevulinate synthase genomic and cDNA clones have permitted the development of expression systems, which have tremendously increased the yields of purified enzyme, facilitating structural and functional studies. A lysine residue has been identified as the residue involved in the Schiff base linkage of the pyridoxal 5-phosphate cofactor, and the catalytic domain has been assigned to the C-terminus of the enzyme. A conserved glycine-rich motif, common to all aminolevulinate synthases, has been proposed to be at the pyridoxal 5phosphate-binding site. A heme-regulatory motif, present in the presequences of 5-aminolevulinate synthase precursors, has been shown to mediate the inhibition of the mitochondrial import of the precursor proteins in the presence of heme. Finally, the regulatory mechanisms, exerted by an iron-responsive element binding protein, during the translation of erythroid 5-aminolevulinate synthase mRNA, are discussed in relation to heme biosynthesis.  相似文献   

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5-Aminolevulinate synthase, a pyridoxal 5'-phosphate-dependent enzyme of the alpha-oxoamine synthase family, catalyzes the first step of the heme biosynthetic pathway in mammalian cells. This reaction entails the condensation of glycine with succinyl-coenzyme A to yield 5-aminolevulinate, carbon dioxide and CoA. Mutations in the erythroid aminolevulinate synthase gene lead to a defective enzyme and are associated with the erythropoietic disorder X-linked sideroblastic anemia. In the past few years, rapid scanning-stopped-flow spectroscopy and chemical quenched-flow studies of the ALAS reaction, under single- and multi-turnover conditions, have provided important results for the interpretation of the catalytic mechanism. In particular, the role of the protein scaffold in modulating the chemical reactivity of the pyridoxal 5'-phosphate cofactor and, thus, the catalytic pathway of ALAS has been investigated in our laboratory using transient kinetics and global analysis of the kinetic data.  相似文献   

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The possibility that glutathione-S-transferases can serve as heme carriers in cells was studied via the following two characteristics: the ability to bind hemin reversibly and the coordination between heme and glutathione-S-transferases level in the cell. two erythroleukemic cell lines that can be induced to synthesize hemoglobin were studied, K-562 and Friend murine erythroleukemia cells. It was found that hemin-associated glutathione-S-transferase tends to lose its native structure as expressed by partial irreversible inhibition of glutathione conjugation activity. In K-562 cells, a small increase in heme synthesis was induced, but under no condition could glutathione-S-transferase be elevated. In addition, introduction of high hemin from without caused large hemoglobin production but did not induce changes in the glutathione-S-transferase content. Dimethyl sulfoxide-induced Friend murine erythroleukemia cells synthesized a large amount of endogenous hemin that had to be transported from the mitochondria for hemoglobin synthesis. Although a concomitant increase in glutathione-S-transferase level (20-40%) was observed, it was only short-lived, unlike hemin, which continued to increase. These data indicate a lack of correlation between glutathione-S-transferase and hemin or hemoglobin levels. Finally, dimethyl sulfoxide-induced cells were treated with succinyl acetone to inhibit heme synthesis. These cells showed the same increased levels and time-dependent pattern of glutathione-S-transferase as untreated cells. A similar phenomenon was observed when different substrates were used to measure the activities of glutathione-S-transferases. These results raise doubts about the possibility of glutathione-S-transferases functioning as heme carriers in cells.  相似文献   

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