RESUMEN
The role of Ser-308 of murine D-aspartate oxidase (mDASPO), particularly its side chain hydroxyl group, was investigated through the use of site-specific mutational analysis of Ser-308. Recombinant mDASPO carrying a substitution of Gly, Ala, or Tyr for Ser-308 was generated, and fused to either His (His-mDASPO), or glutathione S-transferase, His, and S (GHS-mDASPO) at its N-terminus. Wild-type His-mDASPO or GHS-mDASPO or their mutant derivatives were expressed in Escherichia coli and purified by affinity chromatography. All purified recombinant proteins had functional DASPO activity. The Gly-308 and Ala-308 mutants had significantly higher catalytic efficiency towards D-Asp and N-methyl-D-Asp, and a higher affinity for flavin adenine dinucleotide (FAD) compared to the wild-type enzyme. The Tyr-308 mutant had lower catalytic efficiency and binding capacity. These results suggest that the side chain hydroxyl group of a critical residue of mDASPO, Ser-308, down-regulates enzymatic activity, substrate binding, and FAD binding. This study provides information on the active site of DASPO that will considerably enhance our understanding of the biological significance of this enzyme.
Asunto(s)
Sustitución de Aminoácidos , D-Aspartato Oxidasa/química , Serina/química , Animales , Sitios de Unión/genética , Catálisis , D-Aspartato Oxidasa/genética , D-Aspartato Oxidasa/metabolismo , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/genéticaRESUMEN
The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT-PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.
Asunto(s)
Sustitución de Aminoácidos , D-Aspartato Oxidasa/química , D-Aspartato Oxidasa/genética , Sistemas de Lectura Abierta , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Escherichia coli/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Especificidad por Sustrato/genética , PorcinosRESUMEN
In our previous reports [Z. Long, H. Homma, J.-A. Lee, T. Fukushima, T. Santa, T. Iwatsubo, R. Yamada, K. Imai, FEBS Lett. 434 (1998) 231-235; Z. Long, M. Sekine, M. Adachi, T. Furuchi, K. Imai, N. Nimura, H. Homma, Arch. Biochem. Biophys. 404 (2002) 92-97], we demonstrated for the first time that D-aspartate (D-Asp) is actually synthesized in cultured mammalian cells such as PC12, MPT1, and GH3 cells. After its synthesis, this unique amino acid is spontaneously and continuously released into the extracellular space during cell culture. In the current study, we characterized two different types of D-Asp efflux in PC12 cells. One is a spontaneous and continuous form of release of cytoplasmic origin that does not involve exocytotic efflux of vesicular origin. Endogenous D-Asp is predominantly localized to the cytoplasm of cells, and this form of D-Asp release presents a striking contrast to exocytotic, quantal discharge of vesicular dopamine. The other form of efflux is also of cytoplasmic origin and occurs through volume-sensitive organic anion channels that are opened upon hyposmotic stimuli. Interestingly, this latter form of efflux is potentiated by acetylcholine stimulation.
Asunto(s)
Citosol/metabolismo , Ácido D-Aspártico/metabolismo , Acetilcolina/farmacología , Animales , Transporte Biológico Activo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Vesículas Citoplasmáticas/metabolismo , Dopamina/metabolismo , Exocitosis , Activación del Canal Iónico , Nifedipino/farmacología , Concentración Osmolar , Células PC12 , Ratas , Proteínas SNARE/fisiologíaRESUMEN
In an attempt to identify genes that can confer resistance to cisplatin, we introduced a yeast genomic library into Saccharomyces cerevisiae and selected for transformants that grew in the presence of a normally toxic concentration of cisplatin. Plasmids were rescued from the transformants and were analyzed for the presence of individual open reading frames that conferred resistance to cisplatin. We isolated two genes, CIN5 and YDR259c, that increased resistance to cisplatin when overexpressed in Saccharomyces cerevisiae. These genes encoded two proteins, Cin5 and Ydr259c, that were homologous to yAP-1, a basic leucine zipper transcriptional factor that is known to mediate cellular resistance to various toxic agents. The two proteins exhibited stronger homology to each other (33.2% identity, 49.2% similarity) than to all other gene products in S. cerevisiae. Overexpression of each of these proteins also conferred resistance to two DNA-alkylating agents, methylmethanesulfonate and mitomycin C. An experiment with fusion proteins with green fluorescent protein revealed that Cin5 and Ydr259c were localized constitutively in the nuclei of yeast cells. Our results suggest that Cin5 and Ydr259c might be involved in pleiotropic drug-resistance and might protect yeast against the toxicity of cisplatin and other alkylating agents via a single mechanism. These two nuclear proteins might act as transcriptional factors, regulating the expression of certain genes that confer resistance to DNA-alkylating agents.
Asunto(s)
Cisplatino/farmacología , Proteínas Nucleares/fisiología , Proteínas Recombinantes de Fusión , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efectos de los fármacos , Factores de Transcripción , Alquilantes/farmacología , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Farmacorresistencia Microbiana/genética , Farmacorresistencia Microbiana/fisiología , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The removal of cholesterol from macrophages is important for reversing foam cell formation. In a previous study, we demonstrated that mouse peritoneal macrophages in culture secrete significant amounts of unesterified cholesterol from the lysosomes into the medium during endocytosis and subsequent metabolism of cholesterol-containing liposomes [Furuchi, T., Aikawa, K., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 27345-27348]. In this study, we found that at least two distinct mechanisms are involved in this process. The efflux of unesterified cholesterol into the medium was greatly suppressed by pregnenolone, an inhibitor of lysosomal cholesterol transport, but an appreciable proportion of the unesterified cholesterol was still released into the medium. Analysis of the medium containing the secreted cholesterol by NaBr density gradient ultracentrifugation revealed that the unesterified cholesterol was distributed in two different density peaks (bottom and d =/ approximately 1.1). The d =/ approximately 1.1 peak material formed high-density lipoprotein (HDL)-like particles that were produced and secreted by the macrophages. The lipid components of these particles were phosphatidylcholine and sphingomyelin, while the sole protein component was apolipoprotein E (apo E). Treatment with pregnenolone completely abolished the production of these HDL-like particles but had little effect on the bottom fractions. These data indicate that macrophages release lysosomal cholesterol via both pregnenolone-sensitive and -insensitive pathways, and that only the cholesterol secreted through the pregnenolone-sensitive pathway is associated with endogenously synthesized apo E-containing HDL-like particles. Moreover, we found that the pregnenolone-sensitive pathway operated independently of the presence or absence of exogenous HDL, whereas secretion via the pregnenolone-insensitive pathway was greatly stimulated by exogenously added HDL.
Asunto(s)
Colesterol/metabolismo , Lisosomas/metabolismo , Macrófagos Peritoneales/metabolismo , Animales , Células Cultivadas , Centrifugación por Gradiente de Densidad , Ésteres del Colesterol/metabolismo , Endocitosis , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Ratones , Ratones Endogámicos ICR , Fosfolípidos/metabolismo , Pregnenolona/farmacologíaRESUMEN
Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity. GFA1 encodes L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine-6-phosphate. Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to p-chloromercuribenzoate, a GFAT inhibitor. The cytotoxicity of methylmercury was inhibited by loading excess glucosamine 6-phosphate into yeast. Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts. This report is the first to identify the target molecule of methylmercury toxicity in eukaryotic cells.
Asunto(s)
Glutamato-Amoníaco Ligasa/metabolismo , Compuestos de Metilmercurio/toxicidad , Saccharomyces cerevisiae/efectos de los fármacos , Catálisis , Glutamato-Amoníaco Ligasa/genética , Compuestos de Metilmercurio/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
To identify novel genes that confer resistance to methylmercury (MeHg), a yeast genomic DNA library was transfected into Saccharomyces cerevisiae. Two functional plasmids were isolated from transfected yeast clones D1 and H5 that exhibited resistance to MeHg. The yeast transfected with plasmid isolated from clone H5 was several-fold more resistant than yeast transfected with plasmid from clone D1. Functional characterization of the genomic DNA fragment obtained from clone H5 determined that the GFA1 gene conferred resistance to MeHg. GFA1 was reported to encode L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) which catalyzes the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate. Accumulation of mercury in yeast clone W303B/pGFA1, which contains the transfected GFA1 gene, did not differ from that in control yeast clone W303B/pYES2. The W303B/pGFA1 strain did not show resistance to mercuric chloride, zinc chloride, cadmium chloride or copper chloride, suggesting that the resistance acquired by GFA1 gene transfection might be specific to MeHg. This is the first report of a gene involved in MeHg resistance in eukaryotic cells identified by screening a DNA library.
Asunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/biosíntesis , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Compuestos de Metilmercurio/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , ADN de Hongos/genética , Farmacorresistencia Microbiana , Activación Enzimática/efectos de los fármacos , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Genes Fúngicos/efectos de los fármacos , Vectores Genéticos/genética , Biblioteca Genómica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Compuestos de Metilmercurio/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , TransfecciónRESUMEN
Cultured macrophages take up and metabolize cholesterol-containing liposomes, resulting in massive accumulation of cholesteryl esters in the cells. Using this system, the effects of azole antimycotics on cholesteryl ester formation were studied. Incubation of mouse peritoneal macrophages with ketoconazole, miconazole, or econazole (0.1-10 microM) resulted in concentration-dependent inhibition of cholesteryl ester synthesis from endocytosed cholesterol. IC50 values (concentration resulting in 50% inhibition) were 1.4 +/- 0.1 microM, 4.1 +/- 0.2 microM, and 3.6 +/- 0.2 microM for ketoconazole, miconazole, and econazole, respectively. Complete inhibition was observed with 10 microM ketoconazole, and miconazole and econazole, each at 10 microM, caused 70 and 75% inhibition, respectively, of cholesteryl ester synthesis. The mechanism underlying the inhibition by ketoconazole was further studied. Ketoconazole did not appreciably block the uptake of liposomes or formation of triacylglycerol up to 10 microM. Interestingly, ketoconazole suppressed only 30% of 25-hydroxycholesterol-induced endogenous cholesterol esterification under conditions where esterification of endocytosed cholesterol was completely inhibited. Cytochemical studies with filipin-cholesterol staining revealed that ketoconazole induced massive accumulation of endocytosed cholesterol in macrophage phagolysosomes. These results indicate that ketoconazole inhibits cholesteryl ester formation in macrophages by blocking the intracellular transport of endocytosed cholesterol from lysosomes to the endoplasmic reticulum.
Asunto(s)
Antifúngicos/farmacología , Ésteres del Colesterol/metabolismo , Cetoconazol/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Animales , Antifúngicos/química , Azoles/química , Azoles/farmacología , Células Cultivadas , Colesterol/metabolismo , Econazol/farmacología , Endocitosis , Esterificación , Femenino , Hidroxicolesteroles/metabolismo , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/fisiología , Ratones , Ratones Endogámicos ICR , Miconazol/farmacologíaRESUMEN
Previously we showed that activation of Erk in quiescent cells occurs in the caveolae fraction isolated from fibroblasts. Since the structure and function of caveolae is sensitive to the amount of cholesterol in the membrane, it might be that a direct link exists between the concentration of membrane cholesterol and mitogen-activated protein (MAP) kinase activation. We acutely lowered the cholesterol level of the caveolae fraction by incubating Rat-1 cells in the presence of either cyclodextrin or progesterone. Cholesterol-depleted caveolae had a reduced amount of several key protein components of the MAP kinase complex, including Ras, Grb2, Erk2, and Src. Incubation of these cells in the presence of epidermal growth factor (EGF) caused a rapid loss of EGF receptor from the caveolae fraction, but the usual recruitment of c-Raf was markedly inhibited. Despite the reduced amount of c-Raf and Erk2 in the cholesterol-depleted caveolae fraction, EGF caused a hyperactivation of the remaining caveolae Erk isoenzymes. This was followed by an increase in the amount of active Erk in the cytoplasm. The increased amount of activated Erk produced under these conditions was linked to a 2-fold higher level of EGF-stimulated DNA synthesis. Even cholesterol depletion by itself stimulated Erk activation and DNA synthesis. These results suggest that the MAP kinase pathway can connect the cholesterol level of caveolae membrane to the control of cell division.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Colesterol/metabolismo , Endotelio Vascular/metabolismo , Animales , Línea Celular , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , RatasRESUMEN
During normal spermatogenesis, more than half of the germ cells undergo apoptosis, but the physiological significance and molecular mechanisms of this programmed cell death are largely unknown. Because Bcl-2 functions as a death repressor, we have investigated the effect of misexpressing Bcl-2 in spermatogonia in transgenic mice using the human bcl-2 cDNA under the control of the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter. In the 2-week-old transgenic testes, exogenous Bcl-2 was expressed in spermatogonia and massive accumulation of spermatogonia was observed in seminiferous tubules by 4 weeks. At this time, only a few spermatocytes were apparent, and the accumulated cells degenerated, leading to vacuolization in some seminiferous tubules by 7 weeks. In older transgenic mice, abnormal accumulation of spermatogonia and degeneration of these germ cells was still observed, but some seminiferous tubules in which the level of Bcl-2 expression was reduced recovered normal spermatogenesis. These observations indicate that spermatogonial apoptosis is part of the normal program of mammalian spermatogenesis and is regulated by a pathway affected by Bcl-2.
Asunto(s)
Apoptosis/fisiología , Proteínas Proto-Oncogénicas/fisiología , Espermatogénesis/fisiología , Espermatogonias/citología , Testículo/citología , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Expresión Génica , Humanos , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Espermatogénesis/genéticaRESUMEN
Cultured mouse peritoneal macrophages effectively take up and metabolize liposomes containing phosphatidylserine and cholesterol, resulting in massive accumulation of cholesteryl esters and triacylglycerols in their cytoplasm (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226-5231). With this system, various steroid derivatives were assessed as to their ability to inhibit the cholesteryl ester formation from endocytosed cholesterol in macrophages. Among the steroids tested, one group of steroids having an oxo group at the C17 or C20 position, such as androstenedione, dehydroisoandrosterone, progesterone and pregnenolone, completely inhibited cholesteryl ester formation at 10 microM. Another group of steroids having a hydroxy group at the C17 position, such as testosterone and androstenediol, had a lesser effect; complete inhibition of cholesteryl ester formation was achieved with 100 microM or more. The mechanism underlying the inhibition by the former class of steroids was further studied. These steroids did not block the uptake or lysosomal hydrolysis of liposomes, nor esterification of fatty acyl chains into triacylglycerols. Moreover, dehydroisoandrosterone and pregnenolone, both of which possess a hydroxy group at the C3 position, had essentially no effect on 25-hydroxycholesterol-stimulated esterification of endogenous cellular cholesterol. On the other hand, androstenedione and progesterone, which possess an oxo group at the C3 position, had a mild inhibitory effect on the esterification of endogenous cholesterol. Upon incubation with a steroid having an oxo group at the C17 or C20 position, free cholesterol taken up by macrophages was accumulated in phagolysosomes, as judged from cytochemical study with filipin-cholesterol staining. These results indicate that a series of structurally-related steroids characterized by the presence of an oxo group at the C17 or C20 position inhibit cholesteryl ester formation in macrophages through blocking the intracellular transport of endocytosed cholesterol from lysosomes to endoplasmic reticulum.
Asunto(s)
Colesterol/metabolismo , Lisosomas/metabolismo , Macrófagos Peritoneales/metabolismo , Esteroides/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Ésteres del Colesterol/biosíntesis , Endocitosis , Hidroxicolesteroles/farmacología , Macrófagos Peritoneales/citología , Ratones , Ratones Endogámicos ICR , Esteroides/química , Relación Estructura-Actividad , Triglicéridos/biosíntesisRESUMEN
Certain steroids having an oxo group at the C-17 or C-20 position such as pregnenolone and dehydroisoandrosterone inhibit the cholesterol transport from lysosomes to other cellular sites. Taking advantage of the fact that the inhibition is reversed upon removal of the steroids, we studied the factors that control the cholesterol transport from lysosomes to other cellular sites in macrophages. Macrophages that accumulated unesterified cholesterol in their lysosomes were prepared by incubating cells with liposomes containing cholesterol and phosphatidylserine in the presence of a steroid inhibitor. These cells were chased by means of steroid washout, and then the effects of various pharmacological agents on the subsequent metabolism of cholesterol were examined. When the cells were chased in the absence of the agents, some of the cholesterol was converted to cholesteryl esters in the cells, and others were desorbed into the medium as unesterified forms, suggesting recovery of lysosomal cholesterol trafficking. Among the agents tested, bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, completely blocked both cholesterol esterification and cholesterol desorption at 10 nM. Moreover, agents that neutralize the lysosomal proton gradient, such as ammonium chloride and chloroquine, also reduced both of the processes. Fluorescent microscopic examination of bafilomycin A1-treated cells revealed extensive filipin-cholesterol staining of perinuclear lysosomes. From these data, we conclude that acidic pH is required for the efflux of cholesterol from lysosomes to other cellular sites.
Asunto(s)
Antibacterianos/farmacología , Colesterol/metabolismo , Lisosomas/efectos de los fármacos , Macrólidos , Macrófagos/efectos de los fármacos , ATPasas de Translocación de Protón/antagonistas & inhibidores , Vacuolas/enzimología , Animales , Transporte Biológico , Células Cultivadas , Colchicina/farmacología , Citocalasina B/farmacología , Lisosomas/metabolismo , Macrófagos/metabolismo , Ratones , Pregnenolona/farmacologíaRESUMEN
The nucleotide sequence of one of the putrescine transport operons (pPT71), located at 16 min of the Escherichia coli chromosome, was determined. It contained the genes for an induced ornithine decarboxylase and a putrescine transport protein. The gene for the ornithine decarboxylase contained a 2,196-nucleotide open reading frame encoding a 732-amino acid protein whose calculated Mr was 82,414, and the predicted amino acid sequence from the open reading frame had 65% homology with that of a constitutive ornithine decarboxylase encoded by the gene at 64 min. The ornithine decarboxylase activity was observed in the cells carrying pPT71 cultured at pH 5.2, but not in the cells cultured at pH 7.0. The gene for the putrescine transport protein contained a 1,317-nucleotide open reading frame encoding a 439-amino acid protein whose calculated Mr was 46,494. The hydropathy profile of the putrescine transport protein revealed that it consisted of 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length. The transport protein was in fact found in the membrane fraction. When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of its own promoter, the putrescine transport activity increased greatly. The results suggest that the gene expression of the operon is repressed strongly under standard conditions.
Asunto(s)
Antiportadores , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Ornitina Descarboxilasa/genética , Putrescina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Bacterianos/ultraestructura , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/enzimología , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Peso Molecular , Operón , Mapeo Restrictivo , SolubilidadRESUMEN
The nucleotide sequence of the gene for the spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding A, B, C, and D proteins. By making several subclones, we showed that expression of all the four proteins was necessary for maximal spermidine and putrescine transport activity. A single transport system was involved in the transport of both spermidine and putrescine. The A protein (Mr 43K) was found to be associated with membranes, as shown by Western blot analysis of the cell fractions. In addition, it had consensus amino acid sequences for the nucleotide binding site. B (Mr 31K) and C (Mr 29K) proteins consisted of six putative transmembrane spanning segments linked by hydrophilic segments of variable length as shown by cell localization of the proteins synthesized in maxicells and by hydropathy profiles. D protein (Mr 39K) was inferred to be a polyamine binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. These results indicate that the spermidine and putrescine transport system can be defined as a bacterial periplasmic transport system.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Putrescina/metabolismo , Espermidina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Bacterianos/ultraestructura , Clonación Molecular , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Peso Molecular , Mapeo Restrictivo , Alineación de Secuencia , SolubilidadRESUMEN
Escherichia coli KK313, which was deficient in spermidine transport, was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. E. coli NH1596, which was deficient in spermidine transport and has a 90% decreased putrescine transport activity, was obtained by a second treatment of E. coli KK313 with the same mutagen. Genes for polyamine transport systems were isolated by transforming E. coli NH1596 through DNA fragments from E. coli DR112 using pACYC184 as a vector. One clone for the gene of protein(s) catalyzing both putrescine and spermidine uptake (pPT104) was isolated. Two clones for the genes of protein(s) catalyzing only putrescine uptake (pPT79 and pPT71) were obtained. The genes encoded by pPT104, pPT79, and pPT71 were mapped at 15, 19, and 16 min of E. coli chromosome, respectively. Spermidine uptake by NH1596 carrying pPT104, and by MA261, was not inhibited by putrescine and several polyamine analogues, and the Kt values of these two systems were both approximately 0.1 microM. Putrescine transport by NH1596 carrying pPT104 was inhibited completely by spermidine, N,N-dimethyl-4,4'-bipyridylium (paraquat), and N1-acetyl-spermidine, and the Kt value was 1.4 microM. Putrescine uptake by NH1596 carrying pPT79 or pPT71 was not inhibited by spermidine and several polyamine analogues, and the Kt values were 0.5 and 1.8 microM, respectively. In MA261, the putrescine uptake was inhibited by 25-35% by paraquat and N1-acetyl-polyamines and showed two Kt values, 0.5 and 1.5 microM. Based on these findings, the polyamine transport systems of E. coli are discussed.