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At least 433 records · Page 24Linked to original sources
PubMed1994

Mechanism of prostaglandin E2 release and increase in PGH2/PGE2 isomerase activity by PDGF: involvement of nitric oxide.

We examined the possibility that the platelet-derived growth factor-induced release of prostaglandin E2 and increase in prostaglandin H2 (PGH2)/prostaglandin E2 (PGE2) isomerase activity (EC 5.3.99.3) in NIH3T3 cells was mediated by nitric oxide. Addition of L-NG-nitroarginine methyl ester or diphenyleneiodonium chloride, potent nitric oxide synthase inhibitors, blocks platelet derived growth factor-induced release of prostaglandin E2, lowers basal prostaglandin E2 release, and also blocks the growth factor-induced increase in PGH2/PGE2 isomerase activity. Exogenous nitric oxide stimulates prostaglandin E2 release in NIH3T3 cells and this stimulation is blocked by hemoglobin. In contrast, exogenous nitric oxide failed to induce prostaglandin E2 release from pEJ/ras-transformed cells. The nitric oxide induction of PGH2/PGE2 isomerase activity and prostaglandin E2 release occurred within minutes in contrast to alterations in prostaglandin H synthase/cyclooxygenase. These findings link three different classes of messenger molecules (growth factors, nitric oxide, prostaglandins).

3T3 Cells↗
PubMed1997

Gene cloning and characterization of maleate cis-trans isomerase from Alcaligenes faecalis.

Maleate cis-trans isomerase, which catalyses the conversion of maleate to fumarate, was purified and characterized from Alcaligenes faecalis IFO13111. The molecular weight of maleate isomerase was estimated as 60 kDa, consisting of a 28 kDa dimer as shown by gel-filtration chromatography and SDS-PAGE analysis. Kinetic studies showed that the Michaelis constant for maleate was 4.0 x 10(-5) M. The reverse reaction (fumarate to maleate) activity of the enzyme was detected even though it was quite weak. The maleate isomerase gene (maiA) was cloned by hybridization using the oligonucleotide DNA probes designed on the basis of the determined N-terminal amino acid sequences of the purified enzyme. The determined DNA sequence of the maiA gene contains an open reading frame which encodes a 254-amino-acid sequence. The amino acid sequence of the maiA gene product shows no significant homology to any amino acid sequences in the protein data base.

Alcaligenes↗
PubMed2001

Characterization of a gene encoding a Pichia pastoris protein disulfide isomerase.

Protein disulphide isomerases belong to the thioredoxin superfamily of protein-thiol oxidoreductases that have two double-cysteine redox-active sites and take part in protein folding in the endoplasmic reticulum (ER). We report here the cloning of a Pichia pastoris genomic DNA fragment (2919 bp) that encodes the full length of a protein disulphide isomerase (PpPDI). The deduced amino acid sequence of PDI consists of 517 residues and carries the two characteristic PDI-type redox-active domains -CGHC-, separated by 338 residues, and two potential N-glycosylation sites. The N-terminal end forms a putative signal sequence, and an acidic C-terminal region represents a possible calcium-binding domain. Together with the -HDEL ER retrieval sequence at the C-terminus, these features indicate that the gene encodes a redox-active ER-resident protein disulphide isomerase. The nucleotide sequence, which also contains two other open reading frames, has been submitted to the EMBL Nucleotide Sequence Database, Accession No. AJ302014.

Amino Acid Sequence↗
PubMed1999

Gene structure, chromosomal location, and expression pattern of maleylacetoacetate isomerase.

The gene for maleylacetoacetate isomerase (MAAI) (EC 5.2.1.2) was the last gene in the mammalian phenylalanine/tyrosine catabolic pathway to be cloned. We have isolated the human and murine genes and determined their genomic structure. The human gene spans a genomic region of approximately 10 kb, has 9 exons ranging from 50 to 528 bp in size, and was mapped to 14q24.3-14q31.1 using fluorescence in situ hybridization. The complete catabolic pathway of phenylalanine/tyrosine is normally restricted to liver and kidney, but the maleylacetoacetate isomerase gene is expressed ubiquitously. This suggests a possible second role for the MAAI protein different from phenylalanine/tyrosine catabolism. We have searched for mutations in the maleylacetoacetate isomerase gene in four cases of unexplained severe liver failure in infancy with clinical similarities to hereditary tyrosinemia type I (pseudotyrosinemia). Several amino acid changes were identified, but all were found to retain MAAI activity and thus represent protein polymorphisms. We conclude that MAAI deficiency is not a common cause of the pseudotyrosinemic phenotype.

Alleles↗
PubMed1998

Biosynthesis of D-amino acid-containing peptides: exploring the role of peptide isomerases.

The discovery of D-amino acid residues in a growing number of gene-encoded peptides suggests that such biochemical modifications are more common than initially thought. In fact, the extent to which D-amino acids are incorporated into peptides by multicellular organisms probably has not been fully realized, since routine Edman sequencing does not provide the absolute stereochemistry of amino acid residues. Unless both the D and L isomers of a particular peptide sequence are isolated, D-amino acid-containing peptides are often identified only after synthesis of naturally-occurring peptide fails to yield the desired activity. To date, D-amino acid residues (e.g., alanine, methionine, leucine, isoleucine, phenyl alanine, asparagine, tryptophan and serine) have been identified in peptides from a variety of species, including frogs, snails, clams, lobsters and spiders. While most have a single D-amino acid residue located near their N-termini, an exception is found with omega-Aga IVB. The examples highlighted in this chapter are the result of a unique strategy of multicellular organisms to circumvent stereochemical limitations imposed by the genetic code in an effort to increase molecular diversity. The presence of D-amino acids permits the generation of novel tertiary structure that could not be accessed from L-amino acids alone. Moreover, advantages of increased potency and protease stability are often observed. Our understanding of the biosynthesis of these D-amino acid-containing peptides is still in its infancy. Nevertheless, the discovery of a novel peptide isomerase from the venom of the Agelenopsis aperta spider provides some important clues to explain the incorporation of single D-amino acid residues within a peptide chain. Given its high homology with other serine proteases, the isomerase may represent an opportune mutation in response to evolutionary pressures. Yet, is the isomerase a unique exception or simply the first in a class of enzymes of varying substrate specificity capable of synthesizing D-amino acid-containing peptides? To be sure, much more remains to be explored about the precise timing and mechanism of the isomerization process, in addition to obtaining further structural data on the enzyme itself. Therein lies the continuation of this fascinating story in enzyme biochemistry.

Amino Acid Isomerases↗
PubMed1980

Biochemical characterization of phosphoglucose isomerase and genetic variants from mouse and Drosophila melanogaster.

A simple and unique procedure was developed to purify phosphoglucose isomerase variants from the whole mouse body extracts and Drosophila homogenate. It involved the use of an 8-(6-aminohexyl)-amino-ATP-Sepharose column followed by a preparative isoelectric focusing. In each case, the enzyme in the homogenate was adsorbed by ionic interaction on the ATP-Sepharose column. Substantial purification was achieved by the affinity elution with the substrate-glucose-6-phosphate. Mouse and Drosophila phosphoglucose isomerase as well as the corresponding variants were shown to be dimers of similar molecular weight and to exhibit similar kinetic properties. The isoelectric points for the variants from DBA/2J and C57BL/6J mice were determined to be 8.4 and 8.7 respectively, while they were 6.8 and 6.3 respectively for Drosophila and 4/4 variants. Differential thermal stability was observed for the two mouse variants but not for the Drosophila ones. Amino acid composition analysis was performed for both mouse and Drosophila enzymes. Rabbit antisera for mouse (DBA/2J) and Drosophila (2/2) enzymes were raised. Within each species, complete immunological identity was observed between the variants. The antisera were used to characterize the null mutants of phosphoglucose isomerase identified in the mouse and Drosophila populations. By rocket immunoelectrophoresis, the null allele of the naturally occurring heterozygous null variant of Drosophila was shown to express no cross-reacting materials (CRM).

Amino Acids↗
PubMed1991

A phosphoglucose isomerase gene is involved in the Rag phenotype of the yeast Kluyveromyces lactis.

The rag2 mutant of Kluyveromyces lactis cannot grow on glucose when mitochondrial functions are blocked by various mitochondrial inhibitors, suggesting the presence of a defect in the fermentation pathway. The RAG2 gene has been cloned from a K. lactis genomic library by complementation of the rag2 mutation. The amino acid sequence of the RAG2 protein deduced from the nucleotide sequence of the cloned RAG2 gene shows homology to the sequences of known phosphoglucose isomerases (PGI and PHI). In vivo complementation of the pgi1 mutation in Saccharomyces cerevisiae by the cloned RAG2 gene, together with measurements of specific PGI activities and the detection of PGI proteins, confirm that the RAG2 gene of K. lactis codes for the phosphoglucose isomerase enzyme. Complete loss of PGI activity observed when the coding sequence of RAG2 was disrupted leads us to conclude that RAG2 is the only gene that codes for phosphoglucose isomerase in K. lactis. The RAG2 gene of K. lactis is expressed constitutively, independently of the growth substrates (glycolytic or gluconeogenic). Unlike the pgi1 mutants of S. cerevisiae, the K. lactis rag2 mutants can still grow on glucose, however they do not produce ethanol.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12.

The genes xylA and xylB were cloned together with their promoter region from the chromosome of Klebsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xylA encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M(r) of 49,793). The gene xylB encodes the enzyme xylulokinase (XK or XylB) with a calculated M(r) of 51,783 (483 amino acids). The two genes successfully complemented xyl mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylAKp 5' upstream region in high copy number (but lacking an active xylB gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5' upstream regions of xylA on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.

Aldose-Ketose Isomerases↗
PubMed1986

Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae.

The structural gene PGI1 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PGI1 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PGI1 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 delta haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1 delta strains did not grow in glucose as sole carbon source. On the other hand pgi1 delta/pgi1 delta diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1 delta mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, glucose-6-P is essential in yeasts, and the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.

Chromosome Deletion↗
PubMed1977

[Carbonyl compounds from the reaction of barley isomerase with linoleic acid hydroperoxides. Their development from the 9- or the 13-hydroperoxide-isomer (author's transl)].

UNLABELLED: Hydroperoxid isomerase from barley was incubated with linoleic acid hydroperoxides (LHPO), containing nearly exclusively the 13-LHPO or the 9-LHPO isomer; the volatile reaction products were isolated, concentrated and investigated by means of gas and radio-gaschromatography. Thus it was possible to establish the precursors of the volatile compounds hexanal, 2-trans-heptenal and 2-trans-octenal, which develop during formerly described reactions of isomerase with substrates, containing 9- and 13-LHPO in equal amounts. 13-LHPO was found to be a precursor of hexanal and 2-tr-octenal, while the 9-LHPO isomer in the barley isomerase LHPO breakdown reaction obviously cannot be accepted as precursor of volatile components. The origin of 2-tr-heptenal could not be clarified; it occured neither in the experiments with predominating 9-LHPO nor in those with predominating 13-LHPO. Perhaps 2-tr-heptenal is only produced in the presence of a defined ratio of both isomeric hydroperoxides. ABBREVIATIONS: 13-LHPO = 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid. 9-LHPO = 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid.

Carboxylic Acids↗
PubMed1977

[Hydroperoxide degradation by oat isomerase. Investigation of the reaction mechanism (author's transl)].

Oat isomerase is inhibited by hydroxyoctadecadienoic acids (monohydroxy acids) to a degree comparable with inhibition by linoleic acid hydroperoxides (LHPO). Hydroxy acids seem to combine with the enzyme like LHPO do. In an experiment on LHPO breakdown by isomerase 1-14C-hydroxy acids were added and it was examined whether the epoxyhydroxy acids are formed by an intermolecular or intramolecular mechanism. In this experiment 1-14C-labeled trihydroxy acids were formed; they arise from the hydrolysis of epoxyhydroxyoctadecenoic acids formed on their part by isomerase effected LHPO-breakdown. It was determined that at least 70% of LHPO are converted by intermolecular reaction.

Edible Grain↗
PubMed1991

Cloning and disruption of the yeast C-8 sterol isomerase gene.

The yeast ERG2 gene codes for the C-8 sterol isomerase, an enzyme required for the isomerization of the delta 8 double bond to the delta 7 position in ergosterol biosynthesis. The ERG2 gene was cloned by complementation of a C-8 sterol isomerase mutant strain (erg2). The complementing region of DNA required to restore ergosterol synthesis to erg2 was limited to a 1.0 kb StuI-BglII fragment. In order to determine whether the ERG2 gene was essential for yeast viability, a LEU2 gene was inserted into the NdeI site (made blunt) of this 1.0 kb fragment. Transformation of a wild type diploid strain with the ERG2 substituted DNA resulted in the generation of viable haploids containing the erg2 null allele (erg2-4::Leu2). These results suggest that the C-8 sterol isomerase activity is not essential for yeast cell viability. This disruption represents the second ergosterol biosynthetic gene in the distal portion of the pathway to be disrupted without adversely affecting cell viability.

Cloning, Molecular↗
PubMed1995

Studies on linoleic acid 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis.

Linoleic acid is sequentially converted to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid by the 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis, which is a common pathogen of wheat. The objective of this study was to separate and characterize the two enzyme activities. The isomerase activity was found mainly in the microsomal fraction of the mycelia and the 8R-dioxygenase in the cytosol. The 8R-dioxygenase could be partially purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography or isoelectric focusing. The 8R-dioxygenase was unstable during purification, but it could be stabilized by glutathione, glutathione peroxidase and ethylenediaminetetraacetic acid. Several protease inhibitors reduced the enzyme activity. Gel filtration with Sephacryl S-300 showed that most 8R-dioxygenase activity was eluted with the front with little retention. Isoelectric focusing in the presence of ethylene glycol (20%) indicated an isoelectric point of pl 6.1-6.3. The enzyme was retained on strong anion exchange columns at pH 7.4 and could be eluted with 0.3-0.5 M NaCl. Incubation of the enzyme with 0.1 mM linoleic acid led to partial inactivation, which may indicate product inhibition. Paracetamol and the lipoxygenase inhibitor ICI 230,487 at 30 microM inhibited the 8R-dioxygenase by 44 and 58%, respectively. 8R-hydroperoxy-9Z,12Z-octadecadienoic acid was isolated from incubations of linoleic acid with the partially purified enzyme or with the cytosol in the presence of p-hydroxymercuribenzoate. The hydroperoxide was rapidly converted by the hydroperoxide isomerase in the microsomal fractions to 7S,8S-dihydroxy-9Z,12Z-octadecadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Conversion of mannose to fructose by immobilized mannose isomerase from Pseudomonas cepacia.

The enzyme mannose isomerase (EC 5.3.1.7) catalyzes the isomerization of D-mannose and D-fructose. The conversion of mannose to fructose is the first step in the principal pathway of mannose dissimilation by Pseudomonas cepacia. This enzyme is induced during growth on medium containing mannose to levels three- to four-fold higher than observed during growth on glucose or citrate. Mannose isomerase was purified from extracts of mannose-grown P. cepacia and was efficiently immobilized onto a porous, noncompressible, ceramic support. The performance of the immobilized enzyme, compared with the soluble enzyme, was evaluated under a variety of operating conditions to examine its potential for use in a process for the production of fructose syrups containing a higher proportion of fructose than is currently possible using glucose isomerase.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

Immobilization of glucose isomerase onto granular chicken bone.

Glucose isomerase was immobilized onto granular chicken bone (BIOBONE) by adsorption. The amount of activity bound relative to an equal amount of free enzyme was 32 +/- 1%, with the estimated specific activity decreasing from 11.1 +/- 0.7 to 3.9 +/- 0.5 U/mg protein with immobilization. Compared with the free enzyme, immobilized glucose isomerase showed a threefold increase in the Km for fructose and a fivefold decrease in Vmax. High operating temperatures were possible (greater than 55 degrees C), but continuous use and long-term storage studies showed gradual losses of activity. Both the binding and the activity of the bone-immobilized enzyme were highly resistant to treatments with detergent, ethanol, and KCl. Studies to determine mass transfer limitation effects on immobilized glucose isomerase showed that these were insignificant for this system.

Adhesiveness↗
PubMed2003

Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni.

The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO(2)) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-(13)C]glucose, [U-(13)C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive ( K(i)=180 microM) and uncompetitive ( K'(i)=350 microM) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively ( K(i)=1.3 microM) with a low contribution of uncompetitive inhibition ( K'(i)=13 microM). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 microM) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 microM). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.

Aldehyde-Lyases↗
PubMed1997

Evidence for an isomeric muconolactone isomerase involved in the metabolism of 4-methylmuconolactone by Alcaligenes eutrophus JMP134.

An enzyme specifically induced during 4-methylmuconolactone metabolism by Alcaligenes eutrophus JMP 134 and that exhibited muconolactone isomerizing activity was purified to homogeneity. The enzyme, involved in the isomerization of 3-methylmuconolactone had a high degree of sequence similarity with muconolactone isomerase of Alcaligenes eutrophus JMP 134 and other previously described muconolactone isomerases of the 3-oxoadipate pathway. Kinetic analysis showed that the enzyme has a substrate spectrum and a reaction mechanism similar to those of the muconolactone isomerase, but that it has distinct kinetic properties.

Alcaligenes↗
PubMed2006

The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants.

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a beta-glucuronidase (GUS) gene was transferred into apple cv. 'Holsteiner Cox' via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1-2 g l(-1) mannose in combination with 30 g l(-1) sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l(-1) and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.

Glucuronidase↗