RESUMEN
Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.
Asunto(s)
Aequorina/genética , Proteínas Luminiscentes/genética , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas/métodos , Aequorina/química , Animales , Mediciones Luminiscentes , Proteínas Luminiscentes/químicaRESUMEN
The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas Portadoras/química , Glucosa/análisis , Proteínas de Transporte de Monosacáridos/química , Proteínas de Unión Periplasmáticas , Secuencia de Bases , Proteínas Portadoras/genética , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes , Modelos Moleculares , Proteínas de Transporte de Monosacáridos/genética , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Conformación ProteicaRESUMEN
15-Oxoprostaglandin 13-reductase (PGR) has been purified to apparent homogeneity from pig lung. The enzyme was estimated to have a molecular mass of 36 kDa by both SDS/PAGE and non-denaturing PAGE, indicating that the enzyme is a monomer. 15-Oxo-PGE1, 15-oxo-PGE2 and 15-oxo-PGF2alpha were found to be substrates for the enzyme, whereas the corresponding 15-hydroxyprostaglandins were not. The reverse reaction, the oxidation of 13,14-dihydro-15-oxo-PGE1 to 15-oxo-PGE1, was not observed. Either NADH or NADPH could serve as a coenzyme. However, the Vmax with NADH was approx. 3-fold that with NADPH, while the Km for NADPH was approx. one-tenth that for NADH. Cloning of the cDNA was achieved by PCR and library screening. A 600 bp PCR product containing the sequences of three different tryptic peptides derived from purified PGR was used for cDNA library screening by plaque hybridization. A cDNA clone that contained the entire PGR coding sequence of 987 bp was obtained. The sequence codes for a protein of 329 amino acid residues with a calculated molecular mass of 35791 Da. Homology analysis indicated that the sequence is virtually identical with that of leukotriene B4 (LTB4) 12-hydroxydehydrogenase [Yokomizo, Ogawa, Uozumi, Kume, Izumi and Shimizu (1996) J. Biol. Chem. 271, 2844-2850]. Expression of this cDNA in Escherichia coli resulted in a protein exhibiting both PGR and LTB4 12-hydroxydehydrogenase activities. However, the specific activity of PGR with 15-oxo-PGE1 as a substrate was approx. 300-fold that of LTB4 12-hydroxydehydrogenase. These results indicate that the cloned cDNA codes for a protein with two different enzyme activities, with 15-oxoprostaglandins as the preferred substrates.
Asunto(s)
15-Oxoprostaglandina 13-Reductasa/aislamiento & purificación , Pulmón/enzimología , 15-Oxoprostaglandina 13-Reductasa/metabolismo , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Técnicas Inmunológicas , Cinética , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , PorcinosRESUMEN
The mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) gene and its 5'-flanking region was cloned from a 129 mouse ES bacteriophage P1 genomic library. The gene contains 7 exons and 6 introns and is 11.3 kb in length. The transcription initiation site was mapped at 35 bases upstream from the ATG start codon. The nucleotide sequence of the 1.6 kb promoter region contains two TATA boxes and a number of potential regulatory elements including Sp1, CRE, GRE, AP1, AP2, NF-IL6 and estrogen receptor binding site. Studies of the promoter's activity showed that the first 400 nucleotides of 5'-flanking region efficiently drove the transcription of the luciferase reporter gene in U936 cells upon stimulation with a phorbol ester.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/biosíntesis , Hidroxiprostaglandina Deshidrogenasas/genética , Regiones Promotoras Genéticas , Animales , Bacteriófago P1 , Secuencia de Bases , Sitios de Unión , Codón , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Exones , Biblioteca Genómica , Intrones , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , TATA Box , Transcripción GenéticaRESUMEN
The cDNA for rat NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was cloned from an intestinal cDNA library. The sequence of this cDNA was found to be identical to that of the reverse transcription-polymerase chain reaction (RT-PCR) product obtained using rat lung RNA as a template. The cDNA contains a 798-bp open reading frame that codes for a protein of 266 amino acids (M(r) 28,775) which shares 88.7% identity with the human 15-PGDH and 92.1% identity with the mouse 15-PGDH protein. The regions that are believed to be the NAD+ binding domain and the active site are conserved in the enzymes from the three different species. However, the sequence of the C-terminal 9 amino acids appears to be significantly different. The authenticity of the rat cDNA was confirmed by the expression of an enzymatically active 15-PGDH in E. coli.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The cDNA for mouse NAD+ dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was isolated from a lung cDNA library. The cDNA contains a 798 bp open reading frame that codes for a protein of 266 amino acids (M(r) 28775) which shares 87% identity with the human 15-PGDH protein. The regions that are believed to form the NAD+ binding site and the active site are conserved in the mouse and human enzymes. The authenticity of the mouse cDNA was confirmed by expression of an active 15-PGDH in Escherichia coli. Northern blot analysis demonstrated that 15-PGDH mRNA is expressed primarily in lung, intestine, stomach and liver.
Asunto(s)
ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica , Hidroxiprostaglandina Deshidrogenasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Pulmón/enzimología , Ratones , Datos de Secuencia Molecular , NAD/metabolismo , Especificidad de Órganos , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión , Homología de Secuencia de AminoácidoRESUMEN
Evidence suggests that one or more cysteine residues may be important for the activity of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). All of these four cysteines (Cys 45, Cys 63, Cys 152, and Cys 182) are found in areas which are believed to be important for the functioning of the enzyme. Site-directed mutagenesis was used to examine the role of the four cysteine residues found in 15-PGDH. Each cysteine was individually changed to an alanine and to phenylalanine. The C182A mutant protein was completely inactive, while the other three alanine mutants retained full activity. When all of the cysteines were individually changed to phenylalanine, only the C45F mutant retained full activity. The C63F mutant enzyme retained only about 10% of the wild-type activity while the C152F and C182F mutants were inactive. From these results it appears that only C182 is necessary for enzyme activity. Mutagenesis of Cys 63 and Cys 152 to phenylalanine lends support to the suggestion that these two residues are located in critical parts of the enzyme.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Placenta/enzimología , Secuencia de Bases , Sitios de Unión/genética , Cisteína/química , Cisteína/genética , ADN Complementario/genética , Femenino , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Técnicas In Vitro , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , EmbarazoRESUMEN
Human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a member of the short-chain dehydrogenase family of enzymes. It has been proposed that a highly conserved serine residue (corresponding to serine 138 of 15-PGDH) may be involved in the catalytic mechanism of many of these enzymes. Site-directed mutagenesis was used to change serine 138 of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase to an alanine. The mutant protein was then expressed in E. coli. Western blot analysis indicated that the S138A mutant protein was expressed at levels similar to the wild type enzyme; however, the mutant protein was found to be inactive. These results support the proposed role of this highly conserved serine in enzyme activity.
Asunto(s)
Alanina , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Mutagénesis Sitio-Dirigida , Placenta/enzimología , Serina , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Secuencia Conservada , Escherichia coli/genética , Femenino , Expresión Génica , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
Relatively little is known about how 15-PGDH activity is regulated. Changes in 15-PGDH activity have been reported in response to physiological changes brought about by aging, pregnancy, hormonal changes, hypertension and smoking. In addition a large number of drugs have been shown to affect 15-PGDH activity both in vivo and in vitro. The availability of the 15-PGDH cDNA will be a valuable tool for studying how this enzyme is regulated. Isolation of the genomic DNA with its promoter regions has not yet been reported.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Femenino , Humanos , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/aislamiento & purificación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NAD/metabolismo , Placenta/metabolismo , Embarazo , Prostaglandinas/metabolismoRESUMEN
125I-N6-(N-[6-N-(5-iodo-4-azidosalicyl)-aminohexyl]- aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (125I-N6-I-ASA-AH-NAD+) was synthesized by coupling N6-([6-aminohexyl]-carbamoylmethyl)-NAD+ with 4-azidosalicylic acid N-hydroxysuccinimide ester followed by radioiodination. The utility of 125I-N6-I-ASA-AH-NAD+ as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydrogenase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD+. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD+. These results indicate that 125I-N6-I-ASA-AH-NAD+ can be a specific photoprobe for NAD(+)-linked enzymes.
Asunto(s)
Marcadores de Afinidad/química , Azidas/química , NAD/análogos & derivados , Glutamato Deshidrogenasa/química , Hidroxiprostaglandina Deshidrogenasas/química , Radioisótopos de Yodo , NAD/química , Oxidorreductasas/química , FotoquímicaRESUMEN
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. In this study the cDNA for human placental 15-PGDH was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity and characterized. The N-terminus of the recombinant protein was sequenced and found to be identical with the known amino-acid sequence of 15-PGDH. Determinations of Km and Vmax values for a number of the prostaglandins and NAD+ indicate that the recombinant enzyme does not appear to be kinetically different from the human placental enzyme. Site-directed mutagenesis was used to examine the importance of two residues which are highly conserved in the short-chain dehydrogenases which are known to be related to 15-PGDH. Tyrosine-151 was changed to phenylalanine and serine while lysine-155 was changed to glutamine and leucine. Western blot analysis indicated that the mutant and wild-type proteins were expressed at the similar levels. However, all of the mutant proteins were found to be inactive. These results indicate that both tyrosine-151 and lysine-155 are required for 15-PGDH activity.
Asunto(s)
Escherichia coli/genética , Expresión Génica , Hidroxiprostaglandina Deshidrogenasas/genética , Lisina/genética , Mutagénesis Sitio-Dirigida , NAD/farmacología , Placenta/enzimología , Tirosina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Femenino , Humanos , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Datos de Secuencia Molecular , Prostaglandinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Type 1 15-hydroxyprostaglandin dehydrogenase (PGDH) is the main enzyme responsible for the metabolism of prostaglandin E2 (PGE2) and PGF2 alpha. To examine the possibility that a deficiency of PGDH might contribute to preterm labor, we measured localization of immunoreactive (IR-) PGDH, PGDH mRNA, and PGDH enzyme activity in chorio-decidua, placenta, and amnion in patients after term elective cesarean section (n = 9), after spontaneous vaginal term delivery (n = 10), and at idiopathic preterm labor (PTL) in the absence of infection (< 36 weeks gestation; n = 11). Localization of IR-PGDH was determined in additional specimens of membranes after PTL with infection (n = 13) and without (n = 37). IR-PGDH was localized in syncytiotrophoblast and intermediate trophoblasts in placenta and in the trophoblast layer of extraplacental chorion, but was absent from amnion in all patient groups. In chorion, the number of IR-positive trophoblasts was significantly reduced in the idiopathic PTL group compared to those in the other groups. The relative abundance of PGDH mRNA in the chorio-decidua, but not the placenta, from spontaneous labor and PTL was significantly less than that after cesarean section. PGDH mRNA in chorio-decidua from preterm patients correlated with PGDH enzyme activity. Undetectable or low IR-PGDH in chorionic trophoblasts was also associated with low enzyme activity. These results suggest that there exists a subset of patients that present in PTL because of reduced PGDH expression in chorionic trophoblasts. We suggest that this relative deficiency would allow PGs synthesized in the amnion or chorion to escape metabolism in the chorion and thereby contribute to the stimulus to idiopathic PTL.
Asunto(s)
Membranas Extraembrionarias/química , Membranas Extraembrionarias/enzimología , Hidroxiprostaglandina Deshidrogenasas/análisis , Trabajo de Parto/fisiología , Trabajo de Parto Prematuro/fisiopatología , Placenta/química , Placenta/enzimología , ARN Mensajero/análisis , Amnios/química , Amnios/enzimología , Northern Blotting , Corion/química , Corion/enzimología , Decidua/química , Decidua/enzimología , Femenino , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Inmunohistoquímica , Miometrio/química , Miometrio/enzimología , Embarazo , ARN Mensajero/genéticaRESUMEN
Oxidation of many prostaglandins at C-15 results in the formation of 15-keto metabolites, which have reduced biological activity. This reaction is catalyzed by NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase. Using the photoaffinity analog of NAD+, [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified a peptide in the adenine ring binding domain of the NAD+ binding site of 15-hydroxyprostaglandin dehydrogenase. The specificity of photolabeling was demonstrated by saturation and protection experiments. Saturation of photolabeling was observed at approximately 45-50 microM with an apparent Kd of 8-10 microM. Approximately 90% of photolabeling could be protected by 200 microM NAD+ when the protein was photolyzed in the presence of 10 microM probe. The photolabeled protein was digested with Staphylococcus aureus V8 or chymotrypsin, and the photolabeled peptides were purified by either boronate affinity chromatography or Fe+3 chelate chromatography followed by reverse phase HPLC. The photolabeled peptide region was identified to be Val32-Glu40.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/química , NAD/química , Placenta/enzimología , Adenina/análisis , Marcadores de Afinidad , Secuencia de Aminoácidos , Aminoácidos/análisis , Azidas , Cromatografía de Afinidad , Humanos , Hidroxiesteroide Deshidrogenasas/química , Hidroxiesteroide Deshidrogenasas/metabolismo , Datos de Secuencia Molecular , NAD/análogos & derivados , Oxidación-Reducción , Péptidos/análisis , FotoquímicaRESUMEN
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.
Asunto(s)
Proteínas Bacterianas/biosíntesis , ADN/genética , Escherichia coli/enzimología , Hidroxiprostaglandina Deshidrogenasas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Escherichia coli/genética , Expresión Génica , Hidroxiprostaglandina Deshidrogenasas/genética , Isopropil Tiogalactósido/farmacología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and [35S]methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.
Asunto(s)
Dexametasona/farmacología , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Ésteres del Forbol/farmacología , Esteroides/farmacología , Acetato de Tetradecanoilforbol/farmacología , Western Blotting , Carcinógenos/farmacología , Línea Celular , Dinoprostona/metabolismo , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática , Humanos , Hidroxiprostaglandina Deshidrogenasas/biosíntesis , Hidroxiprostaglandina Deshidrogenasas/aislamiento & purificación , Cinética , Leucemia Eritroblástica Aguda , Metionina/metabolismo , NAD/metabolismo , Radioisótopos de AzufreRESUMEN
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the biological inactivation of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli. Site-directed mutagenesis was used to convert a highly conserved tyrosine at position 151 in 15-PGDH to an alanine. The DNA coding for this alanine mutant 15-PGDH was expressed in E. coli. Western blot analysis indicated that this mutant protein was expressed in amounts comparable to the wild type enzyme in bacteria, however no 15-PGDH activity could be detected. This result indicates that tyrosine 151 in 15-PGDH is essential for activity.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/genética , Tirosina/genética , Alanina/genética , Secuencia de Aminoácidos , Codón , Activación Enzimática , Escherichia coli/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Gestacionales/genéticaRESUMEN
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the oxidation of many prostaglandins at C-15, resulting in a subsequent reduction in their biological activity. We report the isolation of the cDNA for this enzyme. A human placental lambda gt11 cDNA library was screened using polyclonal antibodies prepared against the human placental enzyme. A 2.5-kilobase cDNA containing the entire coding region for the enzyme was isolated. The cDNA encodes for a protein of 266 amino acids with a calculated Mr of 28,975. Identification of the cDNA as that coding for 15-hydroxyprostaglandin dehydrogenase was based on the comparison of the deduced amino acid sequence with the amino acid sequence of two peptides, one from the rabbit lung enzyme and the other from the human placental enzyme. This cDNA hybridizes with two species of poly(A+) RNA isolated from human placenta: one of 3.4 kilobases and the other of 2.0 kilobases. Isolation of the cDNA for 15-hydroxyprostaglandin dehydrogenase should facilitate studies on the structure, function, and regulation of this enzyme.