Polymorphism of erythrocyte glucosephosphate isomerase in sheep

An electrophoretic analysis of glucosephosphate isomerase (GPI) in seven Italian sheep populations suggests that this locus is more polymorphic than previously supposed. The observed phenotype distributions are in agreement with the hypothesis of the existence of three codominant alleles, GPI*F, GPI*S and GPI*N, GPI*S being the most frequent (0.935 ÷ 1.000).     

Three phenotypes of glucosephosphate isomerase in sheep: improved staining recipe

Contrary to results published recently, we observe three, rather than two, phenotypes for the enzyme glucosephosphate isomerase (EC 5.3.1.9) from sheep. The phenotypic electro phoretic patterns conform to the patterns observed for this dimeric enzyme in other species. Genotype frequencies in a flock of South downs do not deviate significantly from those predicted under the assumption of the Hardy-Weinberg equilibrium. A remarkable observation is that the electro phoretically distinct phenotypes of GPI are largely or entirely obliterated by the addition of 1–10 mmol/1 MgCl₂ to the electro phoretic buffers. Modification of the usual staining recipe for GPI result in greater resolution and shorter staining times.     

A technique for detection and relative quantitative analysis of glucosephosphate isomerase isozymes from nanogram tissue samples

An improved method for detecting and measuring the enzyme glucosephosphate isomerase after starch gel electrophoresis is described. Nitrocellulose filters are used in a gel overlay procedure which increases the sensitivity of the staining reaction and provides a simple means for accurate quantitation of the isozyme pattern. This staining technique may have wider application with other gel media and also with other enzymes.This work was supported by the M.R.C. Group in Developmental Neurobiology, McMaster University, Canada.     

Biochemical genetics of a new glucosephosphate isomerase allele (Gpi-1 c) from wild mice

We have found a new allele at the structural locus for glucosephosphate isomerase (called Gpi-1 ^c ) in a population of wild mice. The Gpi-1 ^c allele codes for an enzyme of greater cathodal electrophoretic mobility than either the Gpi-1 ^a or Gpi-1 ^b alleles found in the wild and in the SM/J and C57BL/6J inbred strains. Mice homozygous for Gpi-1 ^c have erythrocyte enzyme activity reduced to 33% of normal levels, altered pH profile, lowered heat stability, and normal K _m 's when compared with SM/J and C57BL/6J mice. The activity of the enzyme in brain, liver, and kidney is not so markedly lowered, although the electrophoretic mobility, pH profile, and heat stability are altered in these tissues. Deficiencies of erythrocyte glucosephosphate isomerase in man, to this level, can cause severe hemolytic anemia. Homozygotes for Gpi-1 ^c show only mild hematological symptoms. The frequency of Gpi-1 ^c in wild populations of mice is discussed and the occurrence of a further rare allele Gpi-1 ^d is reported.This work was supported by M.R.C. grants to Professor R. J. Berry and Dr. H. Kacser, whom we should also like to thank for much help and useful discussion.     

Glucosephosphate isomerase poiymorphism in sheep

Two types of glucosephosphate isomerase (GPI) are described for the first time in sheep. Type T produced a 3-b and pattern on polyacrylamide gel electrophoresis of serum samples, whereas type M produced a pattern of 5–7 bands. When 23 Scottish Halfbred ewes showing type T were mated to 3 Suffolk rams showing type M, their 32 offspring had frequencies of 0.72 and 0.28 for T and M respectively. There was no conclusive evidence that the types were controlled by codominant alleles or by simple Mendelian inheritance and neither was sex-linked. The frequency of the rarer type (M) was sufficiently high to provide conclusive evidence of true enzyme polymorphism.     

Evidence for a triplicate set of glucosephosphate isomerase structural genes in hexaploid wheat

The glucosephosphate isomerase (GPI) zymogram phenotypes of 46 aneuploid derivatives of the cultivar Chinese Spring of hexaploid wheat were determined. Variation was observed among the strains in the relative level of expression of three GPI isozymes. The relationships observed between chromosomal constitution and zymogram phenotype support the hypothesis that the three GPI isozymes are dimers composed of protomers encoded by a minimum of three homoeologous structural genes located one each in the short arms of chromosomes 1A, 1B, and 1D. The relative levels of expression per dose of chromosome arm of the products of the three arms differ in a manner consistent with the presence of a two-fold greater quantity of the product of 1BS than of the product of 1AS and of 1DS, indicating that 1BS may contain duplicate GPI structural genes.     

Long-range mapping of the calcium release channel and glucosephosphate isomerase loci using pulsed-field gel electrophoresis

Long range restriction maps of the calcium release channel ( CRC ) and glucosephosphate isomerase ( GPI ) loci have been constructed using pulsed field electrophoresis, Southern blotting and CRC- and GPI-specific probes. The maps, deduced from the restriction fragments detected by the probes, covered 1.1 and 0.3 Mb respectively and no overlap between the maps of these closely linked loci was detected. The minimal distance between the GPI and CRC loci was estimated to be at least 500 kb.     

Characterization of a porcine variable number tandem repeat sequence specific for the glucosephosphate isomerase locus

A variable number of tandem repeat from a porcine glucosephosphate isomerase intron has been isolated and sequenced. The repeat has a unit size of 39 bp, is highly conserved and is present in at least 14 copies. Flanking sequences show a sequence periodicity of 53-54 bp and some sequence homology to the 39 bp repeat. A considerable part of the genomic DNA has been lost during subcloning and is considered to be deletion prone or refractory to propagation in E. coli. The tandem repeat is locus specific and detects at least six alleles in BamHI digested porcine DNA. No homology to other tandem repeat sequences has been found.     

A partial cDNA clone for porcine glucosephosphate isomerase: isolation, characterization and use in detection of restriction fragment length polymorphisms

A cDNA library for porcine skeletal muscle was established in the vector pBR322. The library was screened with an oligonucleotide probe coding for a hexapeptide from glucosephosphate isomerase (Gpi). A positive clone with an insert of about 450 bp and restriction sites for PstI, BamHI and PvuII was isolated. A 362-bp PstI fragment was sequenced and shown to contain the codons for the hexapeptide as well as the remaining 29 amino acids of this Gpi peptide. The PstI fragment was used to probe pig genomic DNA. The restriction enzymes PvuII and SacI detected a set of polymorphisms with five bands, behaving as a set of insertion/deletion polymorphisms.     

Duplication of the structural gene for glucosephosphate isomerase and phosphogluconate dehydrogenase inScabiosa columbaria and their phylogenetic implications in the dipsacaceae

Zymograms of glucosephosphate isomerase (GPI) and phosphogluconate dehydrogenase (PGD) revealed three isozymes for each enzyme in the plant speciesScabiosa columbaria. Intergenic heterodimers are formed between the polypeptides coded byGpi-1 andGpi-2 and between those coded byPgd-1 andPgd-2, indicating that a GPI and a PGD locus have been duplicated in the past. The ancestral genes assort independently with their duplicated gene, suggesting that the duplications have originated from a process of translocation. Linkage was found only betweenGpi-1 andPgd-2 and betweenGpi-2 andPgd-1, suggesting that the duplicated loci were located on the same translocated chromosomal segment. Both duplications are present in all other examined species ofScabiosa and inCephalaria andKnautia, two other genera of the Dipsacaceae. The generaSuccisa andDipsacus, also belonging to the Dipsacaceae, do not showGpi-1 activity, makingGpi-2 andPgd-1 the most likely ancestral genes. InSuccisa, the isozymes ofGpi-1 andGpi-2 either overlap orGpi-1 has been silenced. The combined results suggest that a chromosomal segment containingGpi-2 andPgd-1 has been translocated before the divergence ofScabiosa, Cephalaria, Knautia, andSuccisa.     

Phosphomannomutase and phosphoglucomutase in developing Cassia corymbosa seeds

Phosphomannomutase and phosphoglucomutase in developing Cassia corymbosa seeds have been completely separated from each other and from glucose phosphate and mannose phosphate isomerases by a series of chromatographic procedures that included affinity elution chromatography. Some properties, including the K_m for d-mannose 1,6-biphosphate with phosphomannomutase, are described. The activities of phosphoglucomutase and phosphomannomutase in some other plant tissues are also compared. The significance of these enzymes and the pathway of galactomannan synthesis are discussed.     

Schistosoma mansoni: Inhibition of glucosephosphate isomerase and glycolysis by sugar phosphates

Glucosephosphate isomerase (EC 5.3.1.9) of Schistosoma mansoni is inhibited competitively by a number of tetrose, pentose, and hexose phosphates with inhibitor constant (K_i) values in the range of 0.5 to 400 μM. The most potent inhibitor is 5-phospho-d-arabinonate which resembles the cis-enediolate transition state intermediate of the reaction. These analogs were also found to be effective inhibitors of the production of lactate from glucose by suitably supplemented worm homogenates. The rank order of potency of inhibition of glycolysis was inversely related to the magnitudes of the K_i values for glucosephosphate isomerase. These K_i values were similar to those previously reported for mammalian glucosephosphate isomerase, suggesting similarities in the steric and electronic characteristics of the active sites of these isofunctional enzymes. This conclusion was further supported by the observed pH dependence of the inhibition by 5-phospho-d-arabinonate. Although glucosephosphate isomerase is not a rate-limiting enzyme of glycolysis, in the conventional sense, its selective inhibition could be of chemotherapeutic importance, in part because of the accumulation in glycolyzing systems of glucose 6-phosphate which is a potent feedback inhibitor of hexokinase.     

Malic enzyme,phosphoglucomutase and phosphoglucose isomerase isozymes inTrichogramma [Hym.: Trichogrammatidae]

ME, PGM and PGI electrophoretic banding patterns in 20 laboratory cultures representing 14 species ofTrichogramma were studied. Variations in PGM were found inT. exiguum, T. marylandense, andT. pretiosum. PGI also showed variation inT. exiguum, T. marylandense, T. minutum, andT. parkeri. However, ME variations were found only inT. pretiosum. Based on progeny analyses, we concluded that ME is a tetramer inTrichogramma with fast and slow alleles at a single locus, and that both PGM and PGI have a single locus and each has four alleles. PGM is a monomer, but PGI is a dimer.

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Résumé Les bandes électrophorétiques de l'enzyme malique, de la P.G.M. et de la P.G.I. ont été étudiées chez 20 souches de laboratoire représentant 14 espèces deTrichogramma. Des variations de la P.G.M. ont été trouvées chezT. exiguum, T. marylandense etT. pretiosum. La. P.G.I. montre aussi des variations chezT. exiguum, T. marylandense, T. minutum etT. parkeri. Par contre, des variations de l'enzyme malique ne sont trouvées que chezT. pretiosum. En nous basant, sur l'analyse de progénitures, nous avons conclu que l'enzyme malique est un tétramère chezTrichogramma comprenant un allèle “lent” et un alléle “rapide”, à un seul locus, et que la P.G.M. et la P.G.I. ont chacune un seul locus à quatre allèles. La P.G.M. est un monomère mais la P.G.I. est un dimère.

     

Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase

Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and T75S, under a wide range of collision energies (40–160 eV). The results established the order of dimer stability as TWT > T75S > Q64E ∼ Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies.     

Restricted domain mobility in the Candida albicans Ess1 prolyl isomerase

Ess1 is a peptidyl prolyl cis/trans isomerase that is required for virulence of the pathogenic fungi Candida albicans and Cryptococcus neoformans. The enzyme isomerizes the phospho-Ser-Pro linkages in the C-terminal domain of RNA polymerase II. Its human homolog, Pin1, has been implicated in a wide range of human diseases, including cancer and Alzheimer's disease. Crystallographic and NMR studies have demonstrated that the sequence linking the catalytic isomerase domain and the substrate binding WW domain of Pin1 is unstructured and that the two domains are only loosely associated in the absence of the substrate. In contrast, the crystal structure of C. albicans Ess1 revealed a highly ordered linker that contains a three turn α-helix and extensive association between the two tightly juxtaposed domains. In part to address the concern that the marked differences in the domain interactions for the human and fungal structures might reflect crystal lattice effects, NMR chemical shift analysis and ¹⁵N relaxation measurements have been employed to confirm that the linker of the fungal protein is highly ordered in solution. With the exception of two loops within the active site of the isomerase domain, the local backbone geometry observed in the crystal structure appears to be well preserved throughout the protein chain. The marked differences in interdomain interactions and linker flexibility between the human and fungal enzymes provide a structural basis for therapeutic targeting of the fungal enzymes.     

Phosphoglucose isomerase and esterase phenotypes in Crassostrea angulata and C. gigas

The phenotype distributions and the allele frequencies of the phosphoglucose isomerase and esterase loci examined in the samples of Crassostrea angulata (Essex, England) and C. gigas (Brittany) do not differ significantly and the two populations as such are indistinguishable. The validity of the species C. angulata is questioned and it is postulated that the two samples may be geographic isolates of the same species, i.e. C. gigas. The hatchery reared population of C. gigas from Conway is distinguishable from the other samples of Crassostrea examined. The lack of phenotype diversity is attributed to founder effects of the small parental stock imported in 1965. The distributions of all phenotypes are in agreement with Hardy-Weinberg expectations. Phosphoglucose isomerase (E.C. 5.3.1.9.) is a dimer governed by at least four alleles in C. angulata and five alleles in C. gigas. The slower (Es-S) zone of the esterase electrophoretogram would appear, in both species to be governed by four alleles at a single locus. There was no esterase banding which was specific to either species of Crassostrea.     

Electrophoretic and heat-stability polymorphism at the phosphoglucomutase (Pgm) locus in natural populations of Drosophila melanogaster

Genetic polymorphism for electrophoretic and heat-sensitive alleles is known at the phosphoglucomutase (Pgm) locus in Drosophila melanogaster. Analysis of the distribution of electrophoretic and thermosensitive (ts) alleles was carried out in natural populations from Canada and West Africa and compared with already known data on Italian populations [Trippa, G., Loverre, A., and Catamo, A. (1976). Nature 260:42]. The data show the existence of five common alleles, Pgm ^1.00,tr, Pgm ^1,00,ts, Pgm ^0.70,ts, Pgm ^1.20,ts, and Pgm ^1.50,tr, and two rare alleles, Pgm ^0.55,ts and Pgm ^1.20,tr. The most frequent allele is always Pgm ^1.00,tr; the second most common allele is always of the ts type. The cumulated frequencies of ts alleles in the populations varies between 11 and 32%. The heat stability polymorphism is present in all populations examined and shows again the uniform geographic pattern that has been found for electrophoretic variation at this locus.This research was partially supported by an operating grant (to G.R.C.) from the Canadian National Science and Engineering Research Council (NSERC).     

An irreversible inhibitor of peptidyl-prolyl cis/trans isomerase Pin1 and evaluation of cytotoxicity

Pin1 (protein interacting with never in mitosis A-1) is a member of the peptidyl prolyl isomerase (PPIase) family, and catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds. Because Pin1 is overexpressed in various cancer cell lines and promotes cell growth, it is considered a target for anticancer agents. Here, we designed and synthesized a covalently binding Pin1 inhibitor (S)-2 to target Pin1’s active site. This compound inhibited Pin1 in protease-coupled assay, and formed a covalent bond with Cys113 of Pin1, as determined by ESI-MS. The acetoxymethyl ester of (S)-2, i.e., 6, suppressed cyclin D1 expression in human prostate cancer PC-3 cells, and exhibited cytotoxicity. Pin1-knockdown experiments indicated that a target for the cytotoxicity of 6 is Pin1.     

Effects of various metabolites on two phosphoglucomutase allozyme activities from Drosophila melanogaster

The effects of various metabolites on the two most common phosphoglucomutase allozymes (PGM^A and PGM^B) in Drosophila melanogaster have been investigated in vitro. 2,3-Diphosphoglycerate (2,3DPG) inhibited PGM^A and PGM^B to the same degree in the presence of 25 µM glucose-1,6-diphosphate (G1, 6P₂). However a higher concentration of G1,6P₂ partially reversed the inhibition of PGM^A exerted by 2,3DPG, so that in the presence of 150 µM G1,6P₂ the inhibition of PGM^A was half that of PGM^B at pH 6.0. Glycerol-3-phosphate (G3P) had no significant effect at pH 7.4 but exerted an activating effect at pH 6.0 which was more pronounced in the case of PGM^B. ATP, citrate, and fructose-1, 6-diphosphate (F1,6P₂) inhibited both PGM^A and PGM^B. The differences found in vitro between these two allozymes can have a significant impact on in vivo function and, therefore, on the maintenance of PGM polymorphism in experimental populations of D. melanogaster studied in the laboratory.     

Observational and experimental studies on the acquisition of Anisakis sp. larvae (nematoda: Ascaridida) by trout in fresh water

In the enclosed fresh-water environmsnt of Hanningfield Reservoir, Essex, England, Anisakis sp. larvae (parasites of marine fish) were found in 55 per cent of 40 brown trout and in 26·2 per cent of 61 rainbow trout. Experimental infection by intubating larvae into the stomach was more successful in brown trout (50·6 per cent recovery rate) than in rainbow trout (27 per cent recovery rate). Some larvae reached the body-cavity as early as 2 h after infection. They penetrated the region between the oesophagus and intestine immediately posterior to the caecal openings. Fewer larvae successfully penetrated the gut wall of brown trout within 24 h at 8°C than at 15 ± 1°C. It appears that the reservoir trout acquired Anisakis by being fed as juveniles on untreated marine fish offal containing live larvae.