Enzyme Phosphoglycerate Kinase Research Articles

Changing kinetic properties of the two-enzyme phosphoglycerate kinase/NADP-linked glyceraldehyde-3-phosphate dehydrogenase couple from pea chloroplasts during photosynthetic induction

The kinetics of the two enzyme phosphoglycerate kinase (EC 2.7.2.3)/NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) couple are negatively cooperative and will also fit a model for two enzymes acting on one substrate. When the chloroplast is illuminated apparent negative cooperativity is reduced; maximal velocity of only one of the two enzymes in the two-enzyme model is increased. Even after light activation the activity of glyceraldehyde-3-phosphate dehydrogenase appears to be too low to support photosynthesis at calculated levels of glycerate-1,3-bisphosphate in isolated chloroplasts (Marques, I.A., Ford, D.M., Muschinek, G. and Anderson, L.E. (1987) Arch. Biochem. Biophys. 252, 458–466). The activity of the coupled reaction is apparently sufficient to support observed rates of CO 2 fixation, which suggests that glycerate-1,3-bisphosphate may be channeled from the kinase to the dehydrogenase in vivo.

Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter

We report the cloning of the mouse pgk-1 gene encoding the somatic cell isoform of the enzyme phosphoglycerate kinase. The gene is contained within a 16-kb region of the X chromosome and is interrupted by at least ten introns. The promoter region of the pgk-1 gene is rich in G and C nucleotides and contains five copies of the hexadeoxynucleotide, GGGCGG, the potential binding site for the Sp1 transcription factor, a CCAAT sequence, but no TATA box. This promoter functions following DNA-mediated transfection into mammalian cells. The promoter of the mouse pgk-1 gene is homologous to the human pgk-1 promoter. A number of conserved motifs in the promoter may indicate a significant role for these sequences in expression of the pgk-1 gene.

Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat-shock.

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25 degrees C to 38 degrees C transient increases of 6-7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rna1.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress. Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 degrees C to 38 degrees C) or severe (25 degrees C to 42 degrees C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures.

The effect of substrates on the inter-domain interactions of the hinge-bending enzyme 3-phosphoglycerate kinase

A hinge-bending mechanism has been proposed for phosphoglycerate kinase, in which the two domains bend about the connecting “swaist& rsquo; region. In partially denaturing concentrations of guanidinium chloride the substrate 3-phosphoglycerate stabilises one domain against denaturation and destabilises the other. The reduction of mutual stabilisation of the two domains on binding substrate indicates a freeing of the hinge to allow the protein to take up other states rather than a directive mechanism. The stabilisation of both domains at higher concentrations of ATP at which the enzyme is inhibited supports this mechanism.

A futile cycle in erythrocyte glycolysis

The storage lesion which limits the shelf life of human blood in blood banking is associated with a metabolic loss of 2,3-diphosphoglycerate and ATP. This metabolic loss is driven by intracellular ATPases which are usually considered to include the ion pumps and the reactions which maintain the discoid shape of the human erythrocyte. Under the acidic conditions of blood storage, the energy-yielding reactions of the glycolytic pathway are restricted at the hexokinase and phosphofructokinase steps. We show here that under such circumstances the enzyme of the diphosphoglycerate shunt, diphosphoglycerate mutase/phosphatase and the glycolytic enzyme phosphoglycerate kinase can form a futile cycle with ATPase activity. This ATPase activity responds to 2-phosphoglycolate which is known to activate both diphosphoglycerate mutase and diphosphoglycerate phosphatase reactions. When the enzymes of the futile cycle are combined with the enzymes of the lower glycolytic pathway in a reconstitution experiment designed to represent conditions within the stored erythrocyte, the futile cycle does provide an ATPase activity which results in the metabolic loss of 2,3-diphosphoglycerate. An isotope incorporation experiment demonstrates that the futile cycle is active in glucose-depleted erythrocytes.

Activity of X-linked genes in stem and differentiated Musmusculus × Muscaroli hybrid cells

Hypoxanthine phosphoribosyltransferase-deficient (HPRT −) F9-derived teratocarcinoma stem cells carrying an SV40 genome (12-16TG cells) were fused with Mus caroli (M. car.) spleen cells, and a stem cell hybrid containing reduced numbers of M. car. chromosomes was isolated (BC6 stem cell). The BC6 cells containing an active X chromosome from each parental cell were induced to differentiate in retinoic acid, and differentiated clones were isolated. Most differentiated clones retained both parental X chromosomes in active form. One differentiated clone, BC6-13, grew equally well in hypoxanthine / aminopterin / thymidine (HAT) selective medium (which requires an active M. car. HPRT (E.C.2.4.2.8) locus) or in 6-thioguanine (6TG, which would require either loss or inactivation of the M. car. HPRT locus). Using cDNA probes for HPRT and phosphoglycerate kinase (PGK) (E.C.2.7.2.3) loci and biochemical assays for HPRT and PGK enzymes, it was shown that BC6-13 cells, whether grown in nonselective medium, HAT medium, or 6TG-containing medium, retain the HPRT and PGK genes of both parental cells, but the M. car. forms of HPRT and PGK were inactivated in cells treated with 6TG. 6-Thioguanine seems to act as an inducer, one effect of which is X chromosome inactivation, which seems to be complete and irreversible as early as 24 h after addition of 6TG to BC6-13 cells.

Tumors Arising From Injection of Cultured Murine Placental Cells Into Normal Animals: Direct Evidence for Placental Origin of Tumors of Two Histologic Types<xref ref-type="fn" rid="FN2">2</xref>

Long-term culture of placentas obtained from the mating of a congenic C3H male mouse carrying a unique electrophoretic variant of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1) and a normal C3H female mouse has resulted in cell lines that have apparently undergone spontaneous malignant transformation in vitro. When injected into normal syngeneic animals, these cell lines have given rise to invasive carcinomas of two distinct histologic types, an adenocarcinoma and a poorly differentiated carcinoma. Both were demonstrated to be of placental origin by the continued presence of the allozyme coded on the paternal X-chromosome. Compared to murine tumors of other etiologies, these cell lines were characterized by a high intracellular alkaline phosphatase concentration.

Development of the immune system

ABSTRACT Some problems relating to the organisation of the haematopoietic stem cell pool may be approached by the analysis of changes in the patterns of mosaicism of X-chromosome linked products in the peripheral blood of female mice. We have used the A and B allozymes of the X-chromosome linked enzyme phosphoglycerate kinase (PGK-1) for this purpose. Estimates of the proportions of the two allozymes have been made in whole blood of female mice bled at intervals for up to two years, and also in granulocyte and lymphocyte populations purified on a fluorescence activated cell sorter. The intramouse variance in the proportions of PGK-1A and PGK-1B was shown to be low, indicating that many stem cells were contributing to haematopoiesis at any one time. In contrast the inter-mouse variance was relatively high, indicating that the founder number for the bone marrow compartment of the haematopoietic system was of the order of 20.

Probable Clonal Development of Foreign-Body-Induced Murine Sarcomas<xref ref-type="fn" rid="FN2">2</xref>

The number of cells from which tumors induced by subcutaneous implantation of foreign bodies develop was studied in BALB/c mice with X-chromosome-inactivation mosaicism. Because only one of the two X-chromosomes is active in XX somatic cells, a female mouse heterozygous at the X-linked phosphoglycerate kinase (PGK) locus for Pgk-1b and Pgk-1a has two types of cells. In one population Pgk-1b is active and B-enzyme is produced, whereas in the other population Pgk-1a is active and A-type enzyme is synthesized. Normal tissues from these mosaic mice display both enzyme types, but a tumor that develops from a single cell exhibits only one of the two PGK enzyme types. Of 11 sarcomas induced by Millipore filters, 8 showed single-enzyme PGK phenotypes in primary tumors and in tissue cultures derived from them. The 3 other primary sarcomas showed double-enzyme phenotypes. However, transplantation of cultured cells from 2 of these sarcomas resulted in tumors with single-enzyme PGK phenotypes. Thus the data provide confirmatory evidence for the suggestion that most foreign-body-induced sarcomas develop clonally or from a relatively few cells.

Oocyte specific regulation of PGK-1 isozyme activity in female germ cells of the mouse

SUMMARYThe expression of electrophoretic variant forms of the X-linked enzyme phosphoglycerate kinase (PGK-1) was examined during the ontogeny of the female germ-line followingX-chromosome reactivation. Non-growing oocytes from foetal and neonatal ovaries of heterozygous females show a higher PGK-1A isozyme activity, a reflection of prior non-randomX-inactivation favouring activity of theX-chromosome carrying thePgk-1aallele andXceclocus. On oocyte growth, the total PGK-1 enzyme activity increases 40–50 fold and the pattern of PGK-1 isozyme expression changes giving an electrophoretic pattern now skewed in favour of the PGK-1B isozymic form. Activity of the PGK-1B isozyme exceeds that of PGK-1A in all growing oocytes with a total activity greater than 0·075 nmol h−1oocyte−1. Mice homozygous for eitherPgk-1allele show similar PGK-1 specific activities in somatic tissues where oneX-chromosome is active, but oocytes ofPgk-lbhomozygotes show a higher specific activity compared to those ofPgk-1ahomozygotes. Thus increased activity of the PGK-1B isozyme relative to the PGK-1A isozyme is specific to growing oocytes.

X-chromosome inactivation mosaicism in the three germ layers and the germ line of the mouse embryo

Electrophoretic variant forms of the X-linked enzyme phosphoglycerate kinase (PGK-1, E.C.2,7,2,3) have been used to examine X-chromosome mosaicism in tissues from 12 1/2-day post coitum heterozygous female mouse embryos. Samples of yolk-sac endoderm, neural ectoderm, heart (mesoderm), liver (endoderm) and germ cells were analysed from each embryo. In all tissues except yolk-sac endoderm, both PGK-1 isozymes were expressed. The extent of covariance among tissues with respect to the PGK-1 isozyme contribution is consistent with all tissues being derived from the same pool of cells after X-inactivation. The covariance among tissues gives an estimate of the size of this pool (47 cells) and places the earliest time of X-inactivation in epiblast cells between 4 1/2 and 5 1/2 days post coitum. From the independent variance among tissues within an individual, the average primordial precursor pool size for the three germ layers and the germ line itself was estimated as 193 cells.

Clonal interaction in tumours

The development of cancer is contingent on the emergence of at least one clone of transformed cells. One method used to investigate whether human tumours are monoclonal depends on the mosaicism in the normal tissues of women heterozygous for the two forms of the enzyme glucose-6-phosphate dehydrogenase (G-6-PD). This mosaicism results from the inactivation of one X chromosome in all somatic cells and should not exist in a monoclonal population. Following the discovery in feral mice of an electrophoretic variant (A) of the X-coded enzyme phosphoglycerate kinase (PGK-1) which differs from the form (B) found in common laboratory mouse strains it was reported that fibrosarcomas induced chemically in hybrids of feral and laboratory-bred mice expressed both enzyme phenotypes, but the conclusion that both were expressed by neoplastic cells was based solely on morphological evidence. The development of histocompatible substrains of mice homozygous for one or other alloenzyme has made it possible to study the clonal composition of tumours under experimental conditions in which the neoplastic status of subpopulations of cells can be verified by transplantation. The experiments we now report, while confirming that murine fibrosarcomas are often pleoclonal, show that the clonal composition may change markedly during tissue culture and on transplantation to congenic hosts. These changes presumably reflect changes in the growth kinetics of differentiating subpopulations of the tumour. Cloned sublines are less readily transplantable than uncloned tumour cell populations, and some sublines are less readily transplantable than others; this suggests that sublines resistant to a host's attack are selected on transplantation or that some sublines require the cooperation of others to survive. We postulate that changes in clonal composition occur also during tumour development, metastasis and recurrence.

Glycogen metabolizing enzymes in brain

Three enzymes, glycogen phosphorylase, glycogen synthase, and phosphoglucomutase were evaluated in subcellular fractions and in brain regions. Also the development of each of these enzymes was evaluated in whole brain homogenates. Each enzyme increased during the first three weeks of post partum in a manner that is similar to the development of glycolytic enzymes during this period. The specific activity of each enzyme in various subcellular fractions indicated that the enzymes were primarily soluble. Also unlike the glycolytic enzyme phosphoglycerate kinase, the glycogen metabolizing enzymes had a lower specific activity in synaptosomes than in particle free supernatant fractions of homogenates. Regarding regional distribution small (less than twofold) but significant differences were seen between different brain areas. An inverse relationship between the glycogen metabolizing enzymes and hexokinase was observed, that is, regions highest in glycogen synthase and glycogen phosphorylase were lowest in hexokinase and regions highest in hexokinase were lowest in the glycogen metabolizing enzymes.

Special proliferative sites are not needed for seeding and proliferation of transfused bone marrow cells in normal syngeneic mice.

The widely held view that transfused bone marrow cells will not proliferate in normal mice, not exposed to irradiation or other forms of bone marrow ablation, was reinvestigated. Forty million bone marrow cells from male donors were given to female recipients on each of 5 consecutive days, 5 to 10 times the number customarily used in the past. When the recipients were examined 2-13 weeks after the last transfusion, donor cells were found to average 16-25% of total marrow cells. Similar percentages of donor cells were found when variants of the enzyme phosphoglycerate kinase determined electrophoretically were used for identification of donor and recipient cells. Evidence is presented that the proportion of donor cells is compatible with a linear dependence on the number of cells transfused over the range tested--i.e., 20-200 million bone marrow cells injected intravenously. Special proliferative sites thus do not appear to be required.

X-linked gene loci and muscular dystrophy.

It is proposed that examination of known X-linked gene products might elucidate the gene loci and genetic defects underlying X-linked muscular dystrophies. On the basis of this hypothesis, kinetic studies were performed on the enzyme phosphoglycerate kinase (E.C. 2.7.2.3.) in blood taken from subjects with Duchenne and Becker muscular dystrophy and from controls. Although no differences were detected between enzyme from any source, it is considered that further study of X-linked gene products is warranted.

Random X-chromosome inactivation in female primordial germ cells in the mouse

The pattern of expression of the two X chromosomes was investigated in pre-meiotic germ cells from 12 1/2-day-old female embryos heterozygous for the variant electrophoretic forms of the X-linked enzyme phosphoglycerate kinase (PGK-1). If such germ cells carry the preferentially active Searle's translocated X chromosome (Lyon, Searle, Ford & Ohno, 1964), then only the Pgk-1 allele on this chromosome is expressed. This confirms Johnston's evidence (1979, 1981) that Pgk-1 expression reflects a single active X chromosome at this time. Extracts of 12 1/2-day germ cells from heterozygous females carrying two normal X chromosomes show both the A and the B forms of PGK; since only one X chromosome in each cell is active, different alleles must be expressed in different cells, suggesting that X-chromosome inactivation is normally random in the germ line. This result makes it unlikely that germ cells are derived from the yolk-sac endoderm where the paternally derived X chromosome is preferentially inactivated. In their pattern of X-chromosome inactivation, germ cells evidently resemble other tissues derived from the epiblast.

Non‐random X‐chromosome expression early in mouse development

AbstractWe have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X‐chromosome‐linked enzyme phosphoglycerate kinase (PGK‐1), in order to investigate the randomness of X‐chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk‐1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk‐1a/Pgk‐1b heterozygous conceptuses, and in the one informative 5 1/2‐day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (Xm), so soon after the time of X‐chromosome inactivation, favors the possibility that the preferential expression of Xm is a consequence of primary non‐random X‐chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK‐1 band, corresponding to the allozyme produced by the Pgk‐1 allele on Xm, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.

Effect of solvents on methylcholanthrene-induced carcinogenesis in mice.

We have shown previously, with X-chromosome linked phosphoglycerate kinase (PGK) cell markers, that the fibrosarcomas induced in mice by subcutaneous injection of 0.2-2.0 mg of methylcholanthrene (MCA) dissolved in olive oil had a multicellular origin. In this study we used a constant dose of MCA injected subcutaneously and compared the effects on tumorigenesis of using benzene or olive oil as the solvents. The benzene and olive oil groups differed in tumor incidence (100% versus 50%), location (muscle versus skin), and type (rhabdomyosarcomas versus fibrosarcomas). Each of the nine olive-oil MCA-induced tumors showed both PGK enzyme types, whereas 13 ot 16 tumors induced by MCA in benzene displayed only a single PGK type. This difference in PGK phenotypes suggests that the MCA-benzene neoplasms arose from one or a few cells. We conclude from these results that the nature of the solvent plays an important role in the type and number of cells from which tumors induced by subcutaneous injection of MCA arise.

Relationship between the parental origin of the X chromosomes, embryonic cell lineage and X chromosome expression in mice.

SUMMARYThe electrophoretic variants of theX-chromosome-linked enzyme phosphoglycerate kinase (PGK-1) have been used to investigate the randomness ofXchromosome expression in the fetus and various extra-embryonic membranes of the mouse conceptus. The amnion shows essentially random expression of the maternally derivedXchromosome (Xm) and the paternally derivedXchromosome (Xp). The parietal endoderm, however, shows exclusive or preferential expression ofXm. The results support the idea that the randomness ofXchromosome expression is correlated with embryonic cell lineage such thatXmis preferentially (perhaps exclusively) expressed in derivatives of the primitive endoderm and trophectoderm but thatXmandXpare randomly expressed in the derivatives of the primitive ectoderm.Experiments involving ovary transplants, embryo transfers or crosses with heterozygous mothers confirm previous findings thatXmis preferentially expressed regardless of theXchromosome expressed in the reproductive tract. Additional experiments show that the preferentially expressedXchromosome in the parietal endoderm and visceral yolk sac endoderm of a normalXmXpconceptus is alwaysXmregardless of grand-parental origin ofXmand regardless of whether the mother is a normalXXfemale or anXOfemale.Xpis, however, expressed in these tissues hiXpOfemale conceptuses. It is argued that a form of chromosome imprinting occurs at each generation to markXmandXpas different and that this difference influences the choice of whichXchromosomes are expressed in each cell lineage.

Red cell metabolic alterations in postnatal life in term infants: possible control mechanisms.

Red cell glycolytic intermediates and enzymes in term infants in the first year of life were correlated with the fetal hemoglobin concentration (%F), intra- and extracellular venous pH, plasma inorganic phosphorus (Pi) and pyruvate kinase (PK) activity. Changes in the non-age-dependent enzymes phosphoglycerate kinase, enolase, and phosphofructokinase correlated most significantly with the postnatal decline in %F (P less than 0.001), not the age of the red cell population, as reflected in PK activity. The age-dependent enzymes, hexokinase and glucose-6-phosphate dehydrogenase, however, correlated well with PK activity (P less than 0.001). The concentration of glucose-6-phosphate did not correlate significantly with the postnatal decline in %F (P greater than 0.05) or PK (P greater than 0.10), but correalted significantly with the plasma Pi concentration (P less than 0.001). "Total triose phosphate" and 2,3-diphosphoglycerate did not correlate with Pi. It appears from these studies that an extracellular factor, Pi alters the pattern of glycolytic intermediates in term infants and that the postnatal changes in phosphoglycerate kinase, enolase, and phosphofructokinase are unique to the "fetal" red cell and reflect passage from fetal to "adult" erythropoiesis.