Genetic differences in glucose phosphate isomerase activity among mouse embryos

We have compared mouse embryos of three heterozygous, congenic genotypes (with high, medium and low levels of oocyte-coded glucose phosphate isomerase (GPI-1) activity respectively) to test whether 1) the survival time of oocyte-coded GPI-1 activity in the early embryo is affected by its activity level in the oocyte and 2) whether embryo-coded GPI-1 is detected earlier in embryos that inherit low levels of oocyte-coded GPI-1. The oocyte-coded GPI-1 was entirely GPI-1A allozyme in the high and medium groups but was the less stable GPI-1C allozyme in the low group. We determined total GPI-1 activity and the ratio of different GPI-1 allozymes in early embryos and calculated the activity of oocyte-coded and embryo-coded GPI-1. In all three groups, the oocyte-coded enzyme activity remained at a more or less constant level for the first 21 1/2 days. Some oocyte-coded GPI-1 remained in 4 1/2 day embryos from the high and medium groups but was gone by 5 1/2 days. Very little remained in 4 1/2 day embryos that inherited low levels of a less stable form of the enzyme (GPI-1C allozyme). Despite a 4- to 5-fold difference in initial oocyte-coded GPI-1 activity, no differences were seen among the three genotypically distinct groups of embryos in the time of activation of the embryonic Gpi-1s genes. The embryo-coded GPI-1 was first detectable in 3 1/2 day compacted morulae in all three groups. The level of oocyte-coded GPI-1, in the high group, when embryo-coded GPI-1 was first detected was higher than the level in the low group at any stage prior to detection of embryo-coded GPI-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental profile of glucose phosphate isomerase allozymes in parthenogenetic and tetraploid mouse embryos.

Glucose phosphate isomerase (GPI) allozymes were compared in eggs and embryos of the mouse strains C57BL/6-JHan (GPI-1BB) and 129/Sv (GPI-1AA) under different experimental conditions. The quantitative differences in eggs of the two strains disappeared by the blastocyst stage at day 4 to 5, both in fertilized and diploid parthenogenetic embryos. The degree of degradation of oocyte-coded enzyme molecules and the activation of the embryonic genome for GPI appeared to be equivalent in parthenogenetic embryos from heterozygous females when only one or other maternal allele type remained in the egg after meiosis. Also in tetraploid embryos, generated by electrofusion of homozygous fertilized eggs from the two strains, both genomes seemed to be activated at the same time at day 4; here, however, the GPI-1BB allozyme remained predominant up to day 6.     

Expression of glucose phosphate isomerase in interspecific hybrid (Mus musculus x Mus caroli) mouse embryos.

Hybrid Mus musculus x Mus caroli embryos were produced by inseminating M. musculus (C57BL/OlaWs) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3 1/2 days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preimplantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 3 1/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 3 1/2-day samples (12 samples of compacted morulae) but were consistently detected at 4 1/2 days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 electrophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele.     

High activity of an unstable form of glucose phosphate isomerase in the mouse

Quantitative electrophoretic studies of the three allozymes of glucose phosphate isomerase (GPI-1) produced byGpi-1s ^a/Gpi-1s^c heterozygous mice revealed two opposing influences on GPI-1 activity. First, the GPI-1 AC heterodimer is less stable than GPI-1 AA but more stable than the GPI-1 CC homodimer. Second, a genetic determinant that maps close to or within theGpi-1s structural gene causes elevated activity of GPI-1 AC and probably also GPI-1 CC dimers. The relative lability of these allozymes masks this elevated activity in some tissues but the effect is probably ubiquitous. The significance of these observations is discussed.This study was begun while JDW was at the MRC Radiobiology Unit and continued at the Department of Obstetrics and Gynaecology of the University of Edinburgh, where it was supported, in part, by a grant from the Moray Endowment Fund.     

Immunochemical differentiation of glucose phosphate isomerase (GPI) allozymes of the mouse

Polyclonal xenoantisera against mouse GPI-1B and GPI-1C were produced in rabbits and analyzed for their ability to recognize allozyme-specific determinants. These studies showed a high degree of serological similarity among the three allozymes of mouse glucose phosphate isomerase (GPI). However, GPI-1B and GPI-1C could be differentiated from GPI-1A as well as GPI-1A and GPI-1B from GPI-1C using quantitative solid-phase immunobinding assays. In addition, polyclonal and monoclonal alloantibodies specific for GPI-1C were produced in BALB/c (Gpi-1a/Gpi-1a) mice. As indicated by immunoblotting data, the allozyme specificity of rabbit antisera and monoclonal alloantibodies against GPI-1C is dependent on the native structure of that allozyme.     

Immortalized mouse cell lines that lack a functional Rev3 gene are hypersensitive to UV irradiation and cisplatin treatment

The catalytic subunit of polymerase zeta is encoded from the Rev3 gene. The enzyme is conserved through eukaryotic evolution and its main function appears to be translesion synthesis (TLS) over damaged bases that stall DNA replication. In non-vertebrate cells, inactivation of polymerase zeta results in a moderate hypersensitivity to DNA damage but no proliferative defect in the absence of exogenous damage. Mouse embryos that lack Rev3 however have a severe growth defect and are aborted at midgestation. This has suggested that polymerase zeta may be involved in vital processes in mammalian cells. Here we describe the establishment of immortalized mouse fibroblast cell lines that lack a functional Rev3 gene. These were established from homozygously Rev3-targeted mouse embryos that were also heterozygously targeted at the p53 locus, but the cell lines lost the wild type p53 allele during transformation. Cell lines in which the Rev3 gene is targeted on both alleles grow more slowly than control lines and the deficiency is also associated with an increased frequency of cells at the G2/M phase of the cell cycle and augmented apoptosis. Targeted cells are hypersensitive to UV irradiation and cisplatin treatment and arrest at the S or G2/M phase of the cell cycle if exposed to these treatments. Thus, although vital for murine embryonic development, polymerase zeta activity is not essential for continuous proliferation of transformed mammalian cells that lack p53. It does, however, appear to play an important role in allowing mammalian cells to tolerate DNA damage.     

A new allele of glucose phosphate isomerase in dogs

Summary. A new variant of red cell glucose phosphate isomerase (GPI), designated GPI-C, was observed in the chow-chow breed of dog. GPI-C could be separated from the previously reported dog GPI variants (A and B), both by starch gel electrophoresis and by isoelectric focusing (pH 3–10). Family data supported the hypothesis that GPI-C is controlled by a third codominant allele ( GPI C ). GPI C occurred as a rare allele in the Dutch chow-chow population.     

Genetic diversity of glucose phosphate isomerase from Entamoeba histolytica

To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucose phosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, II-, XIV, or XIX. Two alleles were obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eight polymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points were identified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolytica and other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterization of recombinant E. histolytica GPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility of three GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patterns that have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.     

A third variant of glucose phosphate isomerase in pigs

A new variant of glucose phosphate isomerase (GPI), also known as phosphohexose isomerase (PHI), was detected in a primitive pig population.     

A new allele of glucose phosphate isomerase in dogs

A new variant of red cell glucose phosphate isomerase (GPI), designated GPI-C, was observed in the chow-chow breed of dog. GPI-C could be separated from the previously reported dog GPI variants (A and B), both by starch gel electrophoresis and by isoelectric focusing (pH 3-10). Family data supported the hypothesis that GPI-C is controlled by a third codominant allele (GPI C). GPI C occurred as a rare allele in the Dutch chow-chow population.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Degradation of functional triose phosphate isomerase protein underlies sugarkill pathology

Triose phosphate isomerase (TPI) deficiency glycolytic enzymopathy is a progressive neurodegenerative condition that remains poorly understood. The disease is caused exclusively by specific missense mutations affecting the TPI protein and clinically features hemolytic anemia, adult-onset neurological impairment, degeneration, and reduced longevity. TPI has a well-characterized role in glycolysis, catalyzing the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P); however, little is known mechanistically about the pathogenesis associated with specific recessive mutations that cause progressive neurodegeneration. Here, we describe key aspects of TPI pathogenesis identified using the TPI(sugarkill) mutation, a Drosophila model of human TPI deficiency. Specifically, we demonstrate that the mutant protein is expressed, capable of forming a homodimer, and is functional. However, the mutant protein is degraded by the 20S proteasome core leading to loss-of-function pathogenesis.     

Nature of the multiple forms of sweet-potato glucose phosphate isomerase.

The previously reported isoenzymes of sweet-potato glucose 6-phosphate isomerase were resolved by DEAE-cellulose chromatography. The multiple forms exhibited identical electrophoretic properties and electrofocused as a single component with an apparent isoelectric pH of 4.0. Chromatographic studies also suggest that the multiple forms do not represent true isoenzymes.