2,3-DPG levels in relation to red cell enzyme activities in rat fetuses and hypoxic newborns

We have measured in red cells from fetal and adult Sprague-Dawley and Wistar rats the activities of phosphofructokinase (PFK), pyruvate kinase (PK) and diphosphoglyceromutase (DPGM) as key enzymes in the regulation of 2,3-diphosphoglycerate (2,3-DPG) levels to gather information on the possible causes of the low concentration of 2,3-DPG in fetal red cells. The most striking differences were seen with regard to PK and DPGM activities. The activity of PK was ten times higher in fetal compared to adult red cells, whereas red cell DPGM activity was absent in fetuses and high in adults. In addition, we studied postnatal changes in red cell PK and DPGM activities as well as in the 2,3-DPG concentration in Sprague-Dawley rats. The concentration of 2,3-DPG and the activity of DPGM in red cells increased to almost the adult value within 2 and 4 weeks after birth, respectively, while the activity of PK decreased concomitantly. The postnatal changes occurred similarly, when newborn rats grew up under conditions of hypoxic hypoxia at 0.46 atm (pO₂=9.2 kPa). Our studies support the hypothesis that postnatal changes in 2,3-DPG levels are due to changes in the activity of certain glycolytic enzymes and that the switch from fetal-type to adult-type red cells follows a genetically determined time course.Part of the material presented in this paper has been communicated at the IXth International Berlin Symposium on Structure and Function of Erythroid Cells, 1980     

High pyruvate kinase activity causes low concentration of 2,3-diphosphoglycerate in fetal rabbit red cells

1. The high oxygen affinity of fetal blood in rabbits is due to a very low concentration of 2,3-diphosphoglycerate (2,3-DPG) in the red cells. In order to gather informations on the factors responsible for this characteristic we have studied synthesis and break-down of 2,3-DPG in fetal and adult rabbit red cells in vitro and examined possible regulative pathways which may lead to the low 2,3-DPG concentration in vivo. 2. Under conditions where 2,3-DPG and 3-phosphoglycerate (3-PGA) accumulate in adult erythrocytes, i.e. in a solution containing inosine, pyruvate and inorganic phosphate, the amount of 2,3-DPG synthetized in fetal red cells was only 40% of the adult value and 3-PGA was not measurable. Upon inhibition of enolase by NaF, however, both 2,3-DPG and 3-PGA increased to a similar extent in fetal and adult red cells. These findings point towards differences in the pyruvate kinase (PK) reaction which is one of the rate limiting steps of glycolysis. Direct measurements revealed an over tenfold higher PK activity in fetal compared to adult red cells. This higher activity of PK will lead to a decreased concentration of 3-PGA with a consecutive fall in 2,3-DPG concentration. 3. Other factors, like a decreased glucose utilization, a decreased activity of 2,3-DPG mutase or an increased 2,3-DPG phosphatase activity could be excluded as a cause for the low 2,3-DPG concentration in fetal red blood cells. The same holds for extraerythrocytic factors like glucose concentration or pH value in fetal blood. 4. During the postnatal development of rabbits the PK activity decreased. 50 days after birth, PK activity was 20% of the fetal value but still somewhat higher than in adult erythrocytes. This change is paralleled by an increase in 2,3-DPG concentration and half saturation oxygen pressure. With respect to the synthesis of 2,3-DPG and ATP, the fetal rabbit red cell is comparable to hereditary high PK activity in human erythrocytes.     

Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum

The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway, and its immunological characteristics in Mycoplasma are not well known. In this study, the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the conversion of NADH to NAD⁺. Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.     

The effect of potassium concentration on glycolysis and pyruvate kinase in high potassium and low potassium possum erythrocytes

Effects of potassium concentrations on glycolytic rate and pyruvate kinase (PK) activity were investigated in high potassium (HK) and low potassium (LK) erythrocytes from the common brushtail possum (Trichosurus velpecula). Increasing concentrations of potassium resulted in an increase in glycolytic rate and PK activity in both HK and LK possum erythrocytes. Our results are similar to those from HK/LK sheep. We conclude that there is a close link between Na-pump and glycolysis in erythrocytes from sheep and possum.     

Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans

Summary The Aspergillus nidulans pyruvate kinase gene was isolated by heterologous hybridization using the corresponding yeast gene as a probe. A 2.9 kb EcoRI/BamHI fragment, which exclusively hybridized to the yeast gene, was subcloned in pBR322. This clone was used to transform an A. nidulans pkiA deletion mutant to PKI⁺. The analysis of transformants with respect to the kind of integration revealed about 80% homologous integration – 55% by a double cross-over event (type III integration), 25% by a single cross-over event (type I integration). Type II transformants (20%) that arise by non-homologous integration have not been further characterized with respect to the sites of integration.A direct correlation between the number of copies of the gene integrated into the genome and the measured pyruvate kinase activity was found after growth on a glycolytic carbon source. From this, it was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region.However, after growth on a gluconeogenic carbon source, the regulation of gene expression was found to be disturbed. On acetate an increase in activity per gene copy (0.2 IU) was found in the transformants, as compared with wild-type levels. It is suggested that the pyruvate kinase gene is regulated by negative control, and that some sequences involved in this regulation are missing in the cloned fragment.Abbreviations ATP Adenosinetriphosphate - MM Minimal medium - PEP Phosphoenolpyruvate     

The mitogen-activated/extracellular signal-regulated kinase kinase 1/2 inhibitor U0126 induces glial fibrillary acidic protein expression and reduces the proliferation and migration of C6 glioma cells

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.     

Triggering of human natural killer cells through CD16 induces tyrosine phosphorylation of the p72syk kinase

Activation of protein tyrosine kinases (PTK) is an initial and obligatory event for the triggering of human natural killer (NK) cells to cytotoxicity. Of the different PTK detected in NK cells, only p56^lck has previously been shown to participate in NK cell activation. Here we present evidence that another PTK, p72^syk, is involved in activation of NK cells. Stimulation with a monoclonal antibody to the FcγRIII receptor (CD16) induced an increased tyrosine phosphorylation of p72^syk. This phosphorylation correlated with an increased tyrosine kinase activity of p72^syk towards a synthetic peptide substrate. A severalfold increase in the catalytic activity of p72^syk was also seen after treatment of NK cells with an inhibitor of phosphotyrosine phosphatases, pervanadate. We conclude that triggering of the cytotoxic response in NK cells is associated with activation of p72^syk.     

Phylogenetic analysis of the putative phosphorylation domain in the pyruvate kinase of Bacillus stearothermophilus

All sequenced phosphoenolpyruvate synthases (PPS), pyruvate:phosphate dikinases (PPDK) and enzymes I (EI) of the phosphoenolpyruvate:sugar phosphotransferase system comprise the PEP family. Linked to the C terminus of the sequenced pyruvate kinase from Bacillus stearothermophilus (PKBst) is a domain that is homologous to the putative phosphorylation domains of PEP family enzymes. We report sequence and phylogenetic analyses that lead to the following conclusions: (1) the phosphorylation domain of PKBst was derived from a PPS, late in the evolutionary process, after the divergence of PPSs from PPDKs and EIs; (2) this domain is probably functional in phosphoryl transfer; (3) the C-terminal phosphorylation domain in PKBst probably defines a compact domain in all PEP family proteins that is linked to other domains in these proteins via flexible linkers.     

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: Evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca²⁺ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokin-in-octapeptide (CCK₈) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av) in a suspension of acinar cells. However, in the presence of 1.0 M Staurosporine the stimulatory effect of submaximal concentrations of CCK₈ was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca²⁺]_i,av in cells prestimulated with a submaximal concentration of CCK₈. The data obtained with staurosporine indicate that CCK₈-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca²⁺ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK₈-induced Ca²⁺ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK₈-evoked increase in [Ca²⁺]_i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK₈ at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca²⁺]_i,av evoked by the CCK₈ analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca²⁺]_i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK₈ concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca²⁺]_i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca²⁺ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C. The data presented are in support of a negative-feedback role for protein kinase C in the process of receptor-mediated Ca²⁺ mobilization by a process that involves phosphorylation of the CCK receptor, thereby transforming it from a high-affinity state into a low-affinity state.     

Suppression of Ca2+ oscillations induced by cholecystokinin (CCK) and its analog OPE in rat pancreatic acinar cells by low-level protein kinase C activation without transition of the CCK receptor from a high- to low-affinity state

Cholecystokinin (CCK) analogs, JMV-180 and OPE, release Ca²⁺ from intracellular stores and induce oscillations in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_i), but do not generate a detectable rise in inositol 1,4,5-trisphosphate (InsP ₃) levels. In contrast, high concentrations of CCK elevate InsP ₃, as well [Ca²⁺]_i, to a peak which decreases to near basal levels without oscillations. The mechanisms which underlie inhibition of [Ca²⁺]_i oscillations observed with high CCK concentrations are unclear, but are believed to involve a low-affinity CCK receptor state. Alternately, CCK analogs may be weak partial agonists of the phospholipase C pathway, whereas native CCK, as a full agonist of this pathway, stimulates low levels of protein kinase C (PKC) activity. Preincubation of acini with 1 nM 12 O-tetradecanoyl-phorbol 13-acetate (TPA) for 15 min at 37°C did not affect OPE binding to acini, but abolished OPE-induced (at 1 M) [Ca²⁺]_i oscillations without affecting the initial [Ca²⁺]_i spike. These transformed OPE-induced [Ca²⁺]_i responses mimicked those induced by supramaximal CCK octapeptide (CCK-8) concentrations. Inhibition of [Ca²⁺]_i oscillations by 1 nM TPA was reversed by the PKC inhibitor staurosporine (0.2 M). After [Ca²⁺]_i oscillations were induced with OPE or low concentrations of CCK-8 (20 pM), 1 nM TPA caused a gradual slowing of oscillation frequency over 15–20 min without affecting [Ca²⁺]_i spike amplitude. In contrast, 1 M TPA inhibited OPE binding and caused a more generalized inhibition of OPE- and CCK-evoked Ca²⁺ signals. These data suggest that inhibitory effects of low-level PKC activation on agonist-evoked Ca²⁺ signalling are distinct from the effects of high-level PKC activation by 1 M TPA, and do not require the transition of the CCK receptor from a high-affinity to a low-affinity state.     

Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging

The Saccharomyces cerevisiae Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, plays a key role in mitochondrial function, oxidative stress resistance and chronological lifespan. Isc1p functions upstream of the ceramide-activated protein phosphatase Sit4p through the modulation of ceramide levels. Here, we show that both ceramide and loss of Isc1p lead to the activation of Hog1p, the MAPK of the high osmolarity glycerol (HOG) pathway that is functionally related to mammalian p38 and JNK. The hydrogen peroxide sensitivity and premature aging of isc1Δ cells was partially suppressed by HOG1 deletion. Notably, Hog1p activation mediated the mitochondrial dysfunction and catalase A deficiency associated with oxidative stress sensitivity and premature aging of isc1Δ cells. Downstream of Hog1p, Isc1p deficiency activated the cell wall integrity (CWI) pathway. Deletion of the SLT2 gene, which encodes for the MAPK of the CWI pathway, was lethal in isc1Δ cells and this mutant strain was hypersensitive to cell wall stress. However, the phenotypes of isc1Δ cells were not associated with cell wall defects. Our findings support a role for Hog1p in the regulation of mitochondrial function and suggest that constitutive activation of Hog1p is deleterious for isc1Δ cells under oxidative stress conditions and during chronological aging.