Enzyme Phosphoglycerate Kinase Research Articles

Analysis of the cDNA and encoded protein of the human testis-specific PGK-2 gene.

Because of their unique function, germ cells require unique gene products. Thus, although the glycolytic enzyme phosphoglycerate kinase (PGK) is required in all metabolically active cell types, there are two functional PGK genes in the mammalian genome, one, PGK-1, that is X-linked and ubiquitously expressed in all somatic tissues, and a second, PGK-2, that is autosomal and expressed only in spermatogenic cells. Expression of the PGK-2 gene may function solely to compensate for repressed expression of the PGK-1 gene due to X-chromosome inactivation in spermatocytes. Alternative y, the PGK-2 gene could encode an isozyme with unique characteristics that are beneficial to spermatozoa. We have isolated a cDNA of the human PGK-2 gene and used this as probe to demonstrate that transcription of this gene in spermatocytes and spermatids coincides with a period of repressed transcription of the X-linked PGK-1 gene during spermatogenesis in the human testis. We have also analyzed the amino acid sequence and protein characteristics of the PGK-2 isozyme deduced from this cDNA and compared them with that of the human PGK-1 isozyme to show that known structural and functional motifs are conserved in both proteins. Finally, we have examined the distribution of the PGK-1 and PGK-2 isozymes during spermatogenesis in the mouse to show that while the PGK-2 protein does not appear to possess any unique intracellular localization signal, it is more stable in vivo than the PGK-1 protein.

Cloning of the gene for phosphoglycerate kinase from Schistosoma mansoni and characterization of its gene product

As molecules on the surface or associated with the outer covering (tegument) of Schistosoma mansoni are a major focus as potential vaccine candidates, affinity purified antibodies which are specific to the tegumental antigens were used to immunoscreen a λgt11 S. mansoni cercarial cDNA library. One of the identified clones was found to encode the glycolytic enzyme phosphoglycerate kinase (PGK, EC 2.7.2.3). The 1.5-kb cDNA clone has a single open reading frame encoding 416 amino acids and exhibits over 60% identity to PGKs from a number of eukaryotic species. Recombinant S. mansoni PGK (SmPGK) was overexpressed in Escherichia coli, purified, and shown to have PGK enzyme activity. Native protein affinity purified from S. mansoni adult worms was shown by microsequencing to have the same amino-acid sequence as deduced from the cDNA sequence, thus confirming the cDNA clone we identified encodes S. mansoni phosphoglycerate kinase. Antibodies localize the native SmPGK to various tissues including the tegument of 3-h schistosomula and 42-day adult worms.

Transient and equilibrium kinetic studies on yeast 3-phosphoglycerate kinase. Evidence that an intermediate containing 1,3-bisphosphoglycerate accumulates in the steady state.

The structure of the key glycolytic enzyme 3-phosphoglycerate kinase (PGK) is known in detail, but there is little information on its reaction pathway. We have studied its equilibrium and transient kinetics in the direction of 1,3-bisphosphoglycerate (1,3-bis-P-glycerate) production: ATP + 3-P-glycerate<==>ADP + 1,3-bis-P-glycerate. We devised a sensitive method for following this production. PGK is mixed with 3-P-glycerate and [gamma-32P]ATP in a rapid flow quench apparatus. The reaction mixtures are aged for 4 ms or more and then quenched in acid in which any [1-32P]-1,3-P-glycerate decomposes to 3-P-glycerate and 32Pi, which is determined specifically. The Pi reflects accurately the 1,3-bis-P-glycerate in the original reaction mixture, and the kcat obtained is identical to that obtained by the conventional linked assay method with glyceraldehyde-3-phosphate dehydrogenase. This does not support the postulate of a rapid direct transfer of the 1,3-bis-P-glycerate between the kinase and the dehydrogenase [Srivastava, D. K., & Bernhard, S. A. (1986) Science 234, 1081-1086]. We fitted our data to a simple scheme with the formation of binary complexes, the interconversion of substrates to products via ternary complexes, and the release of products. Because of the high turnover of PGK, the work was carried out under cryoenzymic conditions with 40% ethylene glycol in the buffer. The glycol decreased kcat from 80 to 8.5 s-1 (pH 7.5, 4 degrees C), but the Km for 3-P-glycerate and ATP and the equilibrium constants in the scheme were little affected. We carried out two types of experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Study on the mammalian spermatogenic pathway

The mammalian spermatogenic pathway is a complex process that involves the mitotic proliferation of spermatogonia, the meiotic division of spermatocytes, chromosomal condensation, the production of sperm-specific proteins, and the morphogenic differentiation of spermatids to mature sperm. Elucidating the molecular basis of mammalian spermatogenesis is of great importance, not only because spermatogenesis is a fundamental biological event in the organism but because understanding of this mechanism could lead to the development of new therapeutics to overcome certain genetic diseases. Numerous cytological studies have described, in detail, the dramatic morphological changes of the differentiating spermatogenic cells, however, the molecular basis for many of these steps has yet to be elucidated. What has hampered the progress of such research is the lack of suitable experimental systems. Establishing cultured cell lines of testicular cells has been attempted for many times without much success. We have studied the molecular basis for the mammalian spermatogenic pathway, making use of two unique systems; cell-free transcription reactions in nuclear extracts of animal organs and primary culture of rat testicular cells. Expression of a variety of genes is activated or inactivated as the spermatogenic pathway progresses. The isozyme for glycolytic enzyme phosphoglycerate kinase (PGK) changes in spermatogenic cells from somatic-type PGK-1 to sperm-type PGK-2. We showed that the PGK isozyme switch occurs at the mRNA level during the pachytene spermatocyte stage, suggesting that Pgk-1 transcription ceases and Pgk-2 transcription is instead induced at this stage. The mechanism of the Pgk transcription switch was then investigated by cell-free transcription and DNA transfection experiments. We found that the activation of Pgk-2 transcription in the testis is caused mainly by an Ets family transcription factor named TAP-1, and that the same protein is likely to be involved in the inactivation of the Pgk-1 gene during spermatogenesis. Silencer-like negative regulatory DNA sequences and proteins bound to them seemed to be responsible for the repression of Pgk-1 transcription in the somatic tissues. Throughout the spermatogenic pathway, spermatogenic cells remain in close contact with each other and with somatic Sertoli cells in the seminiferous tubules. Although a number of reports have suggested that Sertoli cells play an important role in spermatogenesis, little is known about the mechanism by which these cells influence spermatogenic cell differentiation.4+ f1p4

The Calvin cycle enzyme phosphoglycerate kinase of Xanthobacter flavus required for autotrophic CO2 fixation is not encoded by the cbb operon

During autotrophic growth of Xanthobacter flavus, energy derived from the oxidation of hydrogen methanol or formate is used to drive the assimilation of CO2 via the Calvin cycle. The genes encoding the Calvin cycle enzymes are organized in the cbb operon, which is expressed only during autotrophic growth. Although it has been established that the transcriptional activator CbbR is required for the expression of the cbb operon, it is unclear whether CbbR is the only factor contributing to the regulation of the cbb operon. This paper describes the isolation of X. flavus mutants which were affected in the regulation of the cbb operon. One of the mutant strains was subject to an enhanced repression of the cbb operon promoter by the gluconeogenic substrate succinate and in addition failed to grow autotrophically. The rate of growth of the X. flavus mutant on succinate-containing medium was lower than that of the wild-type strain, but rates of growth on medium supplemented with gluconate were identical. A genomic library of X. flavus was constructed and was used to complement the mutant strain. The nucleotide sequence of the DNA fragment required to restore autotrophic growth of the X. flavus mutant was determined. One open reading frame that displayed extensive similarities to phosphoglycerate kinase-encoding genes (pgk) was identified. The X. flavus mutant lacked phosphoglycerate kinase activity following growth on gluconate or succinate. Introduction of the pgk gene into the X. flavus mutant partially restored the activity of phosphoglycerate kinase. Induction of the cbb operon of the X. flavus wild-type strain resulted in a simultaneous and parallel increase in the activities of ribulose-1,5-biphosphate carboxylase and phosphoglycerate kinase, whereas the latter activity remained absent in the X. flavus pgk mutant. It is concluded that X. flavus employees a single phosphoglycerate kinase enzyme and this is not encoded within the cbb operon.

Structure of the ADP complex of the 3-phosphoglycerate kinase from Bacillus stearothermophilus at 1.65 A.

The structure of the ADP complex of the enzyme 3-phosphoglycerate kinase (PGK, E.C. 2.7.2.3) from Bacillus stearothermophilus NCA-1503 has been determined by the method of molecular replacement. The structure has been refined to an R factor of 0.16 for all data between 10.0 and 1.65 A resolution, using data collected on the Hendrix-Lentfer imaging plate at the EMBL outstation in Hamburg. The r.m.s. deviations from stereochemical ideality are 0.010 and 0.011 A for bonds and planes, respectively. Although crystallized in the presence of the nucleotide product MgATP, the high-resolution structure reveals the bound nucleotide to be MgADP reflecting the low intrinsic ATPase activity of PGK. Although the two domains of this enzyme are found to be some 4.5 degrees closer together than is found in the yeast and horse-muscle apo-enzyme structures, this structure represents the 'open' rather than the 'closed', catalytically competent form, of the enzyme.

Ribosome-deficient plastids affect transcription of light-induced nuclear genes: genetic evidence for a plastid-derived signal

Transcription of ten nuclear genes was analysed in the albostrians mutant of barley (Hordeum vulgare L.). The lack of plastid ribosomes in white seedlings of this mutant results in a complex alteration of nuclear gene expression at the transcriptional level. We found a strong reduction in the accumulation of mRNAs transcribed from nuclear genes encoding chloroplast enzymes involved in the Calvin cycle, the chlorophyll a/b binding protein, and the cytosolic enzyme nitrate reductase. In contrast, the levels of transcripts of the genes encoding the cytosolic glycolytic enzymes glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase were slightly enhanced. Accumulation of chalcone synthase mRNA even reaches much higher levels in white than in green leaves. Ribosome-deficient plastids were combined by crossing with a nuclear genotype heterozygous for the albostrians allele. Analysis of transcript levels in F1 plants having the same nuclear genotype and differing only with respect to their content of normally developed chloroplasts versus undifferentiated mutant plastids, provided strong genetic evidence for the plastid being the origin of a signal (chain) involved in regulation of nuclear gene expression. Results of run-on transcription in isolated nuclei demonstrated that the plastid signal acts at the level of transcription; it does not interfere with gene regulation in general. Mechanisms triggering nuclear gene expression in response to light operate in white mutant leaves: the very low levels of mRNAs derived from nuclear genes encoding chloroplast proteins and the strongly enhanced level of chalcone synthase mRNA were both light inducible. Also the negative regulation of leaf thionein gene expression by light is observed in white albostrians seedlings.(ABSTRACT TRUNCATED AT 250 WORDS)

Tryptophan phosphorescence as a monitor of the solution structure of phosphoglycerate kinase from yeast

The enzyme phosphoglycerate kinase from yeast possesses two tryptophan residues whose phosphorescence spectrum in low-temperature glasses is resolved into two distinct components with 0-0 vibronic bands centered at 408 and 412.5 nm. The thermal profile of the phosphorescence intensity and lifetime shows that the red (longer wavelength) component is quenched in fluid solutions so that the long-lived phosphorescence observed at ambient temperature in buffer is due entirely to the blue (shorter wavelength) component. The remarkable heterogeneity in flexibility of the two chromophores' sites inferred from the thermal behaviour, when analyzed in terms of the crystallographic structure, allows to make a straightforward assignment of the long-lived emission to internal Trp-333. Because in buffer the phosphorescence is due to only one Trp residue the biphasic nature of the decay reveals the presence of stable, slowly interconverting, conformers with profound differences in the internal fluidity of the C-domain. Further, according to the triplet lifetime, complex formation with substrates affect the protein structure in a very selective way. Thus, while 3-phosphoglycerate has practically no influence on the average lifetime, Mg ATP and Mg ADP increases τ by a factor of 1.9 and 5.3, respectively. The change in lifetime implies a remarkable stiffening of the C-domain which is partly relaxed in ternary complexes with 3-phosphoglycerate. These findings are discussed in terms of ligand-induced “closed” conformations of the protein.

A factor stimulating transcription of the testis-specific Pgk-2 gene recognizes a sequence similar to the binding site for a transcription inhibitor of the somatic-type Pgk-1 gene.

The glycolytic enzyme phosphoglycerate kinase (PGK) consists of two isozymes, somatic-type PGK-1 and testis-specific PGK-2. The isozyme switch from PGK-1 to PGK-2 occurs during spermatogenesis at the mRNA level. The distal upstream region of the gene encoding mouse PGK-2 (Pgk-2) possesses a silencer-like negative cis element. In the present study, a positive cis element located in the proximal upstream region and factor(s) bound to it were analyzed in vitro. Cell-free transcription using nuclear extracts of rat organs demonstrated that the region between nucleotide positions -82 and -64, relative to the most distal transcription initiation site at +1, stimulates transcription in testis extracts. The cis element did not act on the promoter of the thymidine kinase gene, suggesting that it stimulates Pgk-2 transcription in a promoter-specific manner. The cis element bound a nuclear factor(s), which we designated TAP-1. Introducing various base substitutions within the cis element revealed that TAP-1-binding to the element requires the sequence 5'-GGAA-3', which is the binding motif for Ets oncoproteins. We previously reported that TIN-1, a transcription inhibitor of Pgk-1, binds to a sequence similar to the Ets-binding site. The addition of an oligo DNA containing the TIN-1-binding sequence of Pgk-1 prevented TAP-1 from binding to the Pgk-2 cis element, and vice versa. These results suggest that both TIN-1 and TAP-1, which are presumably involved in transcription regulation of the two Pgk genes, recognize DNA sequences related to the Ets-binding motif.

Identification of Candida albicans antigens reactive with immunoglobulin E antibody of human sera.

Candida albicans antigens which reacted with immunoglobulin E (IgE) antibodies of 57 allergic patients were detected by immunoblotting. Of the various antigens, the 175-, 125-, 46-, 43-, and 37-kDa antigenic components reacted most frequently with the patient sera. To purify the major antigens, C. albicans cells were fractionated. The 46-, 43-, and 37-kDa antigens were recovered in cytoplasmic fractions, but the 175- and 125-kDa antigens were not recovered in any fraction. The 46-, 43-, and 37-kDa antigens were purified from cytoplasmic fractions by DEAE and P11 ion-exchange chromatography. Antigens were isolated by cutting bands out of sodium dodecyl sulfate-polyacrylamide gels. The purified components confirmed by immunoblotting were next processed for amino acid sequencing. Parts of the sequences of the 46-, 43-, and 37-kDa antigens had significant levels of homology with Saccharomyces cerevisiae glycolytic enzyme enolase, phosphoglycerate kinase, and aldolase, respectively. Rabbit IgG antibodies prepared against the 46- and 43-kDa antigens strongly cross-reacted with the homologous proteins of S. cerevisiae. However, S. cerevisiae enolase and phosphoglycerate kinase did not cross-react with IgE of patient sera. This result suggests that IgE antibodies against only small parts of their epitopes are elevated in the allergic patients. Since enolase is reported to be a major antigen for systemic candidiasis, this enzyme may be the immunodominant protein in both allergies and fungal infections.

Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse

We have looked for effects of deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in the mouse comparable to non-behavioural consequences of HPRT-deficiency in humans. HPRT-deficient humans show abnormalities in haematopoiesis and, in heterozygotes, there is strong selection in haematopoietic tissues against HPRT-deficient cells arising as a result of X-chromosome inactivation. We have examined two situations in mice in which HPRT- and HPRT+ cells occur in the same individual. First, in chimaeras resulting from the injection of HPRT- embryonal stem cells into HPRT+ blastocysts the fate of HPRT- and HPRT+ cell populations was monitored by their expression of different isozymes of glucose phosphate isomerase and also, in those chimaeras that resulted from injecting the male ES cells into female blastocysts, by in situ hybridisation using a Y-chromosome-specific repetitive DNA probe. There was a small statistically significant selection against the HPRT- population in haematopoietic tissues in both XX in equilibrium with XY and XY in equilibrium with XY chimaeras. Second, in female mice doubly heterozygous for HPRT-deficiency and for an electrophoretic variant of the X-linked enzyme phosphoglycerate kinase, there was a similar small statistically significant selection against the HPRT- population in haematopoietic tissues. While further work is required to establish whether this selection is a consequence of the HPRT mutation, it is clear that any selection against cells in the haematopoietic system as a consequence of HPRT-deficiency is at most small compared with the effect seen in humans. In HPRT-deficient human males surviving beyond the normal age of puberty, there is testicular atrophy. However, we find no effect of HPRT-deficiency on the fertility of either male or female mice. Thus, as with effects on behaviour, the consequences of HPRT-deficiency for haematopoiesis and testis development in the mouse are at most small compared with those in the human. We conclude that the reason for the difference in effects between the two species lies in a difference in purine-related intermediary metabolism per se, rather than in its interaction with brain amine biochemistry.

Glycolytic pathway of the human malaria parasite Plasmodium falciparum: primary sequence analysis of the gene encoding 3-phosphoglycerate kinase and chromosomal mapping studies

We have isolated and characterised the gene ( PGK) encoding the glycolytic enzyme 3-phosphoglycerate kinase (PGK) from the human malaria parasite Plasmodium falciparum. This was achieved using the polymerase chain reaction (PCR) to amplify genomic DNA with primers constructed on the basis of conserved regions identified within PGK molecules of other organisms, and using the PCR product to isolate genomic clones. The gene is present in a single copy, encoding a protein of 416 amino acids (aa). The predicted aa sequence (45.5 kDa) displays approx. 60% identity to both human and yeast PGK molecules, and of the three P. falciparum glycolytic enzymes reported to date, has the greatest sequence identity to the host homologue. All aa residues implicated in substrate and cofactor binding and catalysis are conserved in the malarial PGK molecule, but there are major differences in overall composition, with implications for enzyme stability. In asexual blood-stage parasites, a single mRNA transcript of approx. 2.1 kb is observed. We have mapped the PGK gene to chromosome 9 of the parasite, and a further gene encoding a glycolytic enzyme, aldolase, to chromosome 14.

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha.

Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P2-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P3-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P3 production is a consequence rather than a cause of increasing cytosolic Ca2+. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P2, Ins(1,4,5)P3 and Ins(1,3,4,5)P4, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulated glycolytic flux increases the oxygen uptake rate and aerobic ethanol production, during oxido-reductive growth of Saccharomyces cerevisiae

The influence of overproduction of the glycolytic enzyme 3-phosphoglycerate kinase (PGK) in bakers' yeast Saccharomyces cerevisiae YT6 was investigated in glucose-limited aerobic chemostat experiments. Below the critical value of the dilution rate, the switch point of purely oxidative glucose metabolism to oxido-reductive yeast growth ( D crit), the physiology of the PGK-overproducing mutant showed no differences when compared with the wild type YT6. However, at μ values above the D crit, both the respiration and ethanol production of the mutant were increased. Coincidental higher concentrations of end-products of the reductive glucose metabolism (like ethanol, acetate and glycerol) and metabolic intermediates (pyruvate) were found in the culture fluid of the mutant. The increased specific oxygen uptake rate in the PGK overproducer, as a consequence of the enhanced glycolytic flux and accumulation of pyruvate, shows that the respiratory capacity in the wild type strain is not at its maximal level during oxido-reductive growth. However, its ability to react upon a sudden excess of glucose by an upshift in the D or a glucose pulse, can be the limiting factor causing immediate ethanol and acetate production. The high glycolytic rate compared with the respiratory activity may result in the high reductive response. In this view the coupling of anabolic and catabolic activity is very important. During oxido-reductive growth the maximal PGK yield per mol glucose ( Y psm) is low when compared with purely oxidative growth and is even more decreased by an enhanced reduction activity at the higher dilution rates. This in contrast to the biomass yield per mol ATP ( Y ATP), which is only slightly decreased at D crit and is lower for the overproducing mutant in comparison with the wild type strain YT6.

Consequences of phosphoglycerate kinase overproduction for the growth and physiology of Saccharomyces cerevisiae

The physiological consequences of overproduction of the homologous glycolytic enzyme 3-phosphoglycerate kinase (PGK), integrated in 80 PGK1 gene copies in the genome of Saccharomyces cerevisiae are described. This multiple integration and the strong PGK overproduction (maximum 47% of the total soluble cell protein) do not affect the maximal specific growth rate, but cause 40% reduction of the molar growth yield, compared with that of the wild-type host. The extra energy that is needed for protein overproduction is mainly provided by extra fermentation (respirofermentative growth), but respiration is also elevated compared with the reference strains. The increase in the specific oxygen uptake rate indicates that the respiratory capacity of the yeasts is higher than that in the wild-type host, in which the limited capacity of respiration is generally supposed to be at its maximal level at the critical dilution rate, and is thus responsible for the switch to respirofermentative growth. In a medium PGK1 gene copy integrant (about 25 copies), overproduction of 10%–12% PGK has a stimulating effect on the growth yield and energy efficiency. In these cells the growth benefits of overproduction of the glycolytic enzyme are higher than the disadvantages of extra protein synthesis. The overproduction of PGK has also consequences for the glucose affinity of the yeasts: In the more overproducing strain the K s is increased, compared to its reference strains. Elimination of strong overproducing cells from a glucose-limited chemostat culture is caused by two factors: (a) the excision of the PGK genes from the genome, which is of minor importance for wash-out, but the induction process for this overall decline of overproduction, and (b) the physiological selection process for less overproducing cells, caused by differences in affinity for glucose, most obvious at µ ≈ 1/2µmax. However in batch culture and in a chemostat at low specific growth rates, all the overproducing strains show high genetic stability and constantly provide high PGK quantities.

ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeast glycolytic genesPGKandPYK1

The UAS of the yeast gene encoding the glycolytic enzyme phosphoglycerate kinase (PGK) contains several different sequence elements involved in transcriptional activation. These elements include the activator core sequence, which is bound by the RAP1 protein, and three copies of the pentamer sequence 5' CTTCC 3'. Upstream of the activator core sequence is a region (Yfp), identified as the site of a strong DNA-protein interaction. The Yfp region contains the consensus binding site for the factor ABF1. We have purified the Y protein, which binds to the Yfp region, to homogeneity. The Y protein migrates as a doublet on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 125 KDa. These properties are similar to those of ABF1. ABF1 synthesised in vitro bound strongly to the Yfp region and formed a gel retardation complex of identical mobility to the complex formed by the Y protein. UAS1 of the pyruvate kinase gene (PYK1) promoter contains a RAP1 binding site and single copy of the CTTCC sequence. We have now identified an ABF1 binding site close to the RAP1 binding site and CTTCC sequence in the PYK1 promoter. This site is strongly bound by ABF1 in vitro. The organisation of the PGK and PYK1 UASs is thus similar to each other and to the transcriptional silencer HMR(E) which also contains these sequences.

An NMR analysis of the binding of inhibitors to yeast phosphoglycerate kinase.

The binding of a series of inhibitors to the enzyme phosphoglycerate kinase has been studied using NMR to uncover the binding sites and the effects of binding on the protein conformation. The very effective inhibitor, Suramin, causes the most pronounced changes. The design of inhibitors for mobile proteins is discussed.

DNA binding factors for the CpG-rich island containing the promoter of the human X-linked PGK gene.

The gene coding for the glycolytic enzyme phosphoglycerate kinase (PGK-1) is X-linked in mammals and has a G+C-rich 5' region characteristic of several constitutive genes. Despite the fact that PGK-1 is constitutively expressed, it is transcriptionally regulated in female cells by X chromosome inactivation. To study the expression and regulation of the PGK-1 gene, we have analyzed the binding of trans-acting factors to the 5' region of the PGK-1 gene. We detect at least three distinct binding activities that interact in a sequence-specific manner in vitro with at least six different sites in the 5' region. Two of these binding activities generate DNase I-protected footprints centered approximately 360 bp and 130 bp upstream of the transcription start point. We have examined the promoter specificity of the three binding activities in gel mobility-shift assays by competition with cloned promoter fragments of other genes. None of the binding activities interacts exclusively with X-linked promoters. However, one activity binds preferentially to G+C-rich promoters, and another activity appears to bind preferentially to only two of the promoters tested. Previous studies have demonstrated that one HpaII/MspI site, which is included within a footprinted region observed in this study, is fully methylated in the inactive X chromosome and totally unmethylated on the active X chromosome. Competition studies using synthetic oligonucleotides containing 5-methylcytosine at all CpG sites in this region demonstrate that DNA methylation does not significantly alter the affinity between the corresponding binding activity and this binding site.

Catabolite control of the elevation of PGK mRNA levels by heat shock in Saccharomyces cerevisiae.

Heat shock enhances the very high level of transcription of the phosphoglycerate kinase (PGK) gene in fermentative cultures of Saccharomyces cerevisiae. This response of PGK mRNA levels was not found on gluconeogenic carbon sources, and could be switched on or off subject to availability of fermentable carbon source. The addition of glucose to yeast growing on glycerol resulted in acquisition, within 30-60 min, of the ability to elevate PGK mRNA levels after heat shock. In addition, in aerobic cultures growing on glucose the exhaustion of the medium glucose coincided with a loss of the heat-shock effect on PGK mRNA and a switch-over to slower growth by aerobic respiration. Levels of hsp26 mRNA were analysed during these experiments. Contrasting with this requirement for fermentable catabolite for manifestation of a heat-shock response of PGK mRNA levels, the PGK enzyme was not synthesized at a greater level in heat-shocked fermentative than in gluconeogenic cultures. PGK is one of only a few proteins made efficiently after mild heat shock of yeast. Thus, heat-stress-induced elevation of PGK mRNA levels does not appreciably increase PGK synthesis during exposure to high temperatures and so its role may be to assist cells repressed in mitochondrial function during recovery following a heat shock.