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At least 973 records · Page 54Linked to original sources
PubMed1999

The protein disulphide-isomerase family: unravelling a string of folds.

The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys- tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins, including peptide binding, cell adhesion and perhaps chaperone activities. Attention is now turning to the non-redox-active domains of the PDIs, which may play an important role in all of the known activities of these proteins. Thus the presence of both redox-active and -inactive domains within these proteins portends a complexity of functions differentially accommodated by the various family members.

Carboxypeptidases↗
PubMed1975

The subunit structure of phosphoglucose isomerase from bakers' yeast.

Bakers' yeast phosphoglucose isomerase was studied by both chemical and physical methods to determine its submit structure. Gel filtration in 6 M guanidine HCl as well as acrylamide gel electrophoresis of sodium dodecyl sulfatedentured phosphoglucose isomerase showed two speices corresponding to one-half and one-fourth of the preparative molecular weight of 119,400 determined by equilibrium centrifugation. Further centrifugation studies showed that the enzyme could be completely dissociated to species of 30,000 molecular weight. Peptide maps of tryptic hydrolysates of denatured and chemically modified enzyme showed that the protein is composed of four identical or nearly identical sub-units. The results of amino acid analysis, except half-cystine content, were compatible with identical subunits. The appearent partial specific volume and extinction coefficient were also determined.

Amino Acid Sequence↗
PubMed2000

[Glucose isomerase gene knock-out by denatured double-stranded DNA].

After Genetic background analysis of Streptomyces diastaticus No. 7 strain M1033, the modified conditions of M1033 protoplasts and transformation were established. Replacement plasmid for homologous recombination was also constructed by inserting tsr gene into glucose isomerase gene. The homologous recombination of GI gene in M1033 chromosomes was achieved by using denatured linearized DNA fragments and glucose isomerase deficient strain M1033LJ was obtained. It is basic for introducing mutation into M1033 chromosome and realizing site-directed molecular reformation.

Aldose-Ketose Isomerases↗
PubMed2000

The Conradi-Hünermann-Happle syndrome is caused by mutations in the gene that encodes a 8- 7 sterol isomerase and is biochemically related to the CHILD syndrome.

We here review the clinical and genetic features of the Conradi-Hünermann-Happle syndrome. The disease is characterized by chondrodysplasia punctata, linear ichthyosis, cataract, and short stature. The X-linked dominant mode of inheritance was first recognized by Rudolf Happle in the years 1977 to 1981, who also fully delineated the clinical spectrum of this clinico-genetic entity. In the past, linkage studies had firmly excluded the gene for this syndrome from the Xq28 region, but unfortunately had also failed to clearly map the gene elsewhere on the X-chromosome. Very recently, causative mutations were identified in a large number of patients in the gene for emopamil binding protein. This gene is located on the short arm of Xp11.22-23 and also acts as a D8-D7 sterol isomerase. This enzymatic function plays a crucial role in cholesterol biosynthesis. It is of note that very recent investigations by the Marburg group have disclosed that the CHILD syndrome is likewise caused by a similar metabolic defect, namely a deficiency of a 3b-hydroxysteroid dehydrogenase (NSDHL). In the pathway of cholesterol biosynthesis this enzyme functions "upstream" of D8-D7 sterol isomerase and was shown to underlie the mouse mutant bare patches. Molecular studies in these syndromes now allow us to determine which family members carry the mutation and have already provided evidence in the Conradi-Hünermann-Happle syndrome for both gonadal and somatic mosaicism. As gonadal mosaicism seems to be frequent in this disease, a recurrence risk for further pregnancies has to be considered when dealing with a seemingly sporadic case.

3-Hydroxysteroid Dehydrogenases↗
PubMed1975

Isolation and characterization of tissue-specific isozymes of glucosephosphate isomerase from catfish and conger.

In teleosts glucosephosphate isomerase exists as two tissue-specific isozymes. Most tissues contain the more acidic liver-type isozyme, while white muscle contains the more basic isozyme; and a few tissues contain both the liver- and muscle-type isozymes as well as a hybird. The isozymes were isolated from catfish liver and muscle and from conger muscle and shown to be homogeneous by polyacrylamide gel electrophoresis, isoelectric focusing, analytical ultracentrifugation, and rechromatography. Both isozymes are of molecular weight 132,000 (S020,w = 7.0 S) and composed of two subunits of Mr approximately 65,000. The muscle and liver isozymes were shown to have distinct isoelectric points (catfish liver = 6.2; muscle = 7.0) and amino acid compositions. Tryptic peptide maps, after S-carboxymethylation and carbamylation, revealed several distinct differences in the primary structures of the isozymes. Although the isozymes could also be distinguished on the basis of their stabilities, most of their basic catalytic properties were found to be similar. A conger was obtained which was heterozygous for the variant allele at the muscle-glucosephosphate isomerase locus. A comparison of the variant conger muscle isozyme with the wild type revealed a single altered peptide, suggesting a point mutation. The structure-function studies, as well as the genetic studies, clearly establish that the two types of isozymes are of independent genetic origin.

Alleles↗
PubMed1975

Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

The photoinactivation of the Δ (5)-3-ketosteroid isomerase of Pseudomonas testosteroni in the presence of 3-oxo-4-estren-17β-yl acetate and air is accompanied by destruction of histidine and aspartate (or asparagine). The first order rate constant of photoinactivation of the enzyme is equal, within experimental error, to the first order rate constant for the destruction of a single aspartate (or asparagine) residue and is considerably greater than the first order rate constant for the destruction of a single residue of histidine. When the photolysis is carried out under anaerobic conditions, only aspartic acid (or asparagine) is destroyed as enzyme is inactivated. Both inactivation and aspartate (or asparagine) destruction occur to a greater extent in the absence of oxygen than in its presence. The destruction of histidine, on the other hand, is found to be strictly oxygen-dependent. These results suggest that photochemical modification of a single residue of aspartate (or asparagine) is largely, if not entirely, responsible for photoinactivation of the enzyme under these conditions. When irradiated in the presence of 3-oxo-4-entren-17β-yl acetate, performic acid-oxidized bovine pancreatic ribonuclease does not suffer any detectable destruction of its aspartic or asparaginyl residues but does undergo significant destruction of its histidine residues. These observations suggest that the aspartate (or asparagine) residue modification found with isomerase is an active site-directed photochemical reaction, whereas the modification of histidine may not be.

Amino Acids↗
PubMed2001

Isomerase activity of the C-terminal fructose-6-phosphate binding domain of glucosamine-6-phosphate synthase from Escherichia coli.

The isomerase activity of the C-terminal fructose-6P binding domain (residues 241-608) of glucosamine-6-phosphate synthase from Escherichia coli has been studied. The equilibrium constant of the C-terminal domain k(eq) ([glucose-6P]/[fructose-6-P]) = 5.0. A non-competitive product inhibition of the isomerase activity by the reaction product glucose-6-P has been detected. The existence of more than one binding and reaction sites for the substrate fructose-6P on the molecule of glucosamine-6-phosphate synthase can be expected. The fructose-6P binding domain possibly includes a regulatory site, different from the catalytic center of the enzyme.

Amino Acid Sequence↗
PubMed2002

The intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia.

OBJECTIVE: To investigate the intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia (G. lamblia). METHODS: Total genomic DNA of G. lamblia was extracted and partial fragments of the triose phosphate isomerase (tim) gene were amplified by polymerase chain reaction (PCR). All nucleotide sequences were analyzed by using a phylogenetic analysis, which was constructed with parsimony and Neighbor-joining (N-J) methods. RESULTS: A total of 124 variable sites (23% of all sequences detected) was defined, most of which were found at the silent sites of codons. Two similar phylogenetic trees were constructed, subdividing 16 Giardia isolates into two groups. CONCLUSION: The genetic diversity of G. lamblia appeared to be little affected by factors of both host and geography, while natural-selection played an important role in DNA molecular evolution level of the tim gene. The tim gene may be considered a very useful genetic marker of the population genetic structure of G. lamblia.

Animals↗
PubMed1999

[Correlation of conformational change with enzyme activity of glucose isomerase in denaturants monitored with high performance liquid chromatography].

Glucose isomerase (GI) can catalyze in vitro the isomerization of D-glucose to D-fructose. So it is an extremely important industrial enzyme in the commercial conversion of starch to high fructose syrups. In the previous papers, we have purified and characterized the enzyme from streptomyces diastaticus M1033 of China and obtained the crystal structures by X-ray. In this paper, a method for measurement of the dynamic conformational change procedure of glucose isomerase in various concentrations of denaturants by HPLC has been established. At first the relative molecular mass of GI in solution is measured by HPLC on PROTEIN PAK 300SW (7.5 mm i.d. x 30 cm) column. The relative molecular mass of GI is about 150,000. So GI exists as tetramer in the solution without denaturants. In 0-5 mol/L guanidine hydrochloride, incubated at 30 degrees C for 30 min, GI is gradually dissociated into monomer, and at the same time its activity gradually disappears. In various concentrations of urea and incubation at 30 degrees C for 30 min (or 60 degrees C for 1 h), the results are different from that in guanidine, because the monomers peaks of GI is not found. Only in certain concentrations of urea, the small dimer peaks of GI is found, but the activity of GI significantly disappears. Moreover as the increase of the urea concentration, the retention time of tetramer peak is gradually decreases. From the fluorescence spectra, we found the conformation of GI changed in the solution of urea. So perhaps in urea, the conformation of GI become a little unfolded, and the active region is partly damaged, which makes GI partly inactive. Dissociation into inactive monomers and conformation partly unfolding are all the reason of GI inactivation in denaturants.

Aldose-Ketose Isomerases↗
PubMed1992

Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases.

D-Xylose isomerases are metal-ion (Mn2+, Co2+, Mg2+)-requiring tetrameric enzymes. Both the stoichiometry and the binding constants have been determined by titrating the metal-ion-free enzymes from five organisms (Actinomycetaceae and more divergent bacteria) with the respective metal ions using the enzyme activity as indicator of active complex-formation. The following characteristics have been observed for each specific isomerase: (i) two essential metal ion sites (one structural and one catalytic) exist per subunit; (ii) the metal ion binding at one site does not affect the binding at the other site; (iii) of the four possible configurations E, aE, Eb and aEb, only the double-occupied enzyme is active; (iv) the metal ion activation is a time-dependent process; (v) the dissociation constants for both the structural and catalytic sites may be identical or may differ by one or higher orders of magnitude; (vi) metal ion binding is stronger in the order Mn2+ greater than Co2+ much greater than Mg2+; (vii) pronounced increases in Km values concomitant with decreasing equivalents of metal ion added are only observed in the presence of Mg2+ ions.

Aldose-Ketose Isomerases↗
PubMed2005

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59.

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.

Aldose-Ketose Isomerases↗
PubMed1991

Kinetic studies of Mg(2+)-, Co(2+)- and Mn(2+)-activated D-xylose isomerases.

The kinetic parameters for the interconverting substrates D-xylose in equilibrium D-xylulose and D-glucose in equilibrium D-fructose were determined for several D-xylose isomerases, with Mg2+, Co2+ and Mn2+ as metal ion activators. The Km, kcat. and kcat./Km values are tabulated for the anomeric mixtures (observed parameters) as well as for the respective reactive species, i.e. the alpha-pyranose anomers of D-xylose and D-glucose and the alpha-furanose forms of D-xylulose and D-fructose (real parameters). The real Km values and catalytic efficiencies are more favourable for the ketose sugars (reverse reaction) than for the aldose sugars (forward reaction). Comparisons of the kinetic parameters further support the existence of two distinct groups of D-xylose isomerases. Inhibition constants for the cyclic substrate analogues 5-thio-alpha-D-xylopyranose and alpha-D-xylopyranosyl fluoride and for the acyclic substrate analogue xylitol and its dehydrated form 1,5-anhydroxylitol were determined and are discussed.

Aldose-Ketose Isomerases↗
PubMed1991

Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.

We report here the purification and characterization of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme (PMI-GMP) which catalyzes both the phosphomannose isomerase (PMI) and guanosine 5'-diphospho-D-mannose pyrophosphorylase (GMP) reactions of the Pseudomonas aeruginosa alginate biosynthetic pathway. The PMI and GMP activities co-eluted in the same protein peak through successive fractionation on hydrophobic interaction, ion exchange, and gel filtration chromatography. The purified enzyme migrated as a 56,000 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native protein migrated as a monomer of 54,000 molecular weight upon gel filtration chromatography. The apparent Km for D-mannose 6-phosphate was 3.03 mM, and the Vmax was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent Km values of 20.5 microM and 29.5 microM for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction Vmax (5,680 nmol/min/mg of enzyme) was comparable to the reverse reaction Vmax (5,170 nmol/min/mg of enzyme), and the apparent Km for GDP-D-mannose was determined to be 14.2 microM. Both reactions required Mg2+ activation, but the PMI reaction rate was 4-fold higher with Co2+ as the activator. PMI (but not GMP) activity was sensitive to dithiothreitol, indicating the involvement of disulfide bonds to form a protein structure capable of PMI activity. DNA sequencing of a cloned mutant algA gene from P. aeruginosa revealed that a point mutation at nucleotide 961 greatly decreased the levels of both PMI and GMP in a crude extract.

Alginates↗
PubMed1991

Characterization of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues.

The conversion of 3 beta-hydroxy-5-ene steroids by the enzyme complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is an essential step in the biosynthesis of all classes of hormonal steroids. We report the characterization of two types of cDNA clones encoding rat 3 beta-HSD isolated from a rat ovary lambda gt11 cDNA library with a human 3 beta-HSD cDNA probe. Both type I and type II cDNAs encode proteins of 372 amino acids having 94% homology. Transient expression of the type I and the type II 3 beta-HSD cDNAs in HeLa human cervical carcinoma cells reveals that both proteins possess 3 beta-hydroxysteroid dehydrogenase as well as delta 5-delta 4 isomerase activities for both delta 5-pregnene and delta 5-androstene precursors, although the type I 3 beta-HSD protein is more active than the type II. RNA blot analysis using type I 3 beta-HSD cDNA identifies major mRNA transcripts of 1.7 kilobase in rat ovary, testis, and adrenal poly(A)+ RNA. RNase protection assay using type I- and type II-specific cRNA probes revealed the existence of the two corresponding mRNAs in male and female rat adrenals and gonads as well as in female adipose tissue while only type I mRNA is present in male and female kidney. Moreover, in situ hybridization performed using type-specific labeled 24-mer oligonucleotides confirms that type I is the major mRNA species in the ovary and further indicates that both mRNA species have a similar cellular distribution in the ovarian tissue with the highest level of expression found in corpora lutea. Immunoblot analysis using polyclonal antibodies raised against purified human placental 3 beta-HSD identified a single 42-kDa band in rat ovary, testis, and adrenal, which agrees with the calculated molecular masses of 41,911 and 42,150 daltons for the type I and II proteins, respectively. Determination of 3 beta-HSD enzymatic activity using [14C]pregnenolone and [14C]dehydroepiandrosterone as substrates shows that 3 beta-HSD activity is present not only in the gonads and adrenals of animals of both sexes, but also in many peripheral tissues including adipose tissue, mammary gland, kidney, liver, prostate, seminal vesicle, uterus, skin, brain, heart, thymus, pancreas, lung, and spleen. The present data indicate the existence of two mRNAs encoding rat 3 beta-HSD and their differential tissular distribution in both steroidogenic and peripheral tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1990

[Implication of glucose 6 phosphate isomerase (EC 5.3.1.9) activity in blocking segmentation of the mouse ovum at the 2 cell stage in vitro].

The mouse embryo, when grown in media containing glucose, synthesizes and accumulates glycogen. In certain strains, glucose could be responsible for the 2 cell block; it can be replaced for a short time by glutamine, but with an increase in embryo degeneration. We have tested the hypothesis according to which the problems of glycogen accumulation and the developmental arrest could be due to a metabolic lock involving glucose 6 phosphate isomerase (EC 5.3.1.9). Fructose is located immediately downstream from this metabolic lock. We have exactly replaced the glucose in Whittingham M16 culture medium by fructose: Medium F.M. With this culture medium, Swiss one cell embryos, which normally block in M16 medium (greater than 90%), do not block any longer at the 2 cell stage (11.6%). They can be cultured for 4 days with high rates of blastocyst formation (53.6%). Embryo degeneration (23.6%) is lower than that observed in CZB (31%), and not different from our control degeneration in vivo. Moreover fructose seems to be adequate all through the culture period as there is no need for further addition of metabolites, to obtain blastocysts, as observed for CZB. This demonstrates that a lock at the Glucose phosphate isomerase level is responsible for glycogen accumulation and the 2 cell block in the mouse embryo in vitro.

Animals↗
PubMed1990

Resistance of spores and vegetative cells of Streptomyces thermovulgaris strain 127 and the glucose isomerase produced by them to temperature and glutaraldehyde.

A study was made of the effects of high temperatures and glutaraldehyde on the spores and vegetative cells of Str. thermovulgaris strain 127. It was determined at what treatment times for different temperatures and glutaraldehyde concentrations the spore germination and vegetative cell growth stopped. A high heat stability was found for glucose isomerase, the loss in enzymatic activity increasing with the increase in glutaraldehyde concentration and exposure time. It was determined at what optimum parameters of the combined treatment the growth of the strain stopped and a comparatively high level of glucose isomerase activity was preserved.

Aldehydes↗
PubMed1990

Sequence analysis of D-xylose isomerase gene from Escherichia coli.

Gene coding for E. coli D-xylose isomerase was cloned into the plasmid pWR13 from the plasmid pXO100 containing D-xylose operon. Several subclones harboring DNA fragments of different size obtained by step- by-step shortening with Bal31 nuclease were directly sequenced. The complete nucleotide sequence of the 1.6kb DNA fragment carrying D-xylose isomerase gene was determined. The structural gene of 1320 nucleotides codes for a protein of 440 amino acids. The additional 209 nucleotides at 5' end and 82 nucleotides at 3' end include Shine-Dalgarno ribosome-binding site and the transcriptional termination codon, respectively.

Aldose-Ketose Isomerases↗
PubMed1990

Cloning metabolic pathway genes by complementation in Escherichia coli. Isolation and expression of Plasmodium falciparum glucose phosphate isomerase.

Genetic complementation of an Escherichia coli double mutant was used to isolate and express the gene coding for Plasmodium falciparum glucose phosphate isomerase. The gene contains a 1773-base pair open reading frame, has no introns, and maps to P. falciparum chromosome 14. 34% of the deduced amino acid sequence is identical to human glucose phosphate isomerase, with highest similarity in regions of the proposed active sites. The putative initiation site of translation was determined by deletional and oligonucleotide mediated, site-specific mutageneses. Our data suggest that key metabolic enzymes of Plasmodia can be cloned and expressed in E. coli without prior knowledge of the primary amino acid or nucleic acid structure.

Animals↗