EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation

Culture of bovine zygotes in medium SOFaa supplemented with 100 microM EDTA significantly increased cleavage rates during the first 72 hr of development compared to development in SOFaa. However, continued culture in the presence of EDTA for a further 72 hr (total of 6 days of culture) resulted in significantly reduced development to the morulae/blastocyst and blastocyst stages compared to culture without EDTA. Highest rates of development to the morulae/blastocyst stage (56.5%) and to the blastocyst stage (43.2%) were achieved when zygotes were cultured for 72 hr with EDTA before transfer to medium SOFaa without EDTA. Resultant blastocysts also had significantly increased blastocyst cell number and ICM cell number compared to those cultured without EDTA in the first 72 hr. EDTA was shown to inhibit glycolytic activity of the cleavage stage embryo, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for the later stage embryo as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development.     

A mutation in the 3-phosphoglycerate kinase gene allows anaerobic growth of Bacillus subtilis in the absence of ResE kinase

The Bacillus subtilis ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to pgk, the gene encoding 3-phosphoglycerate kinase, failed to suppress a resD mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The pgk-1 mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1, 3-diphosphoglycerate, is responsible for the observed effect of the pgk-1 mutation on anaerobiosis of resE mutant cells.     

Novel role of 3-phosphoglycerate kinase,a glycolytic enzyme,in the activation of L-nucleoside analogs,a new class of anticancer and antiviral agents

l-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of l-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of l-nucleoside analogs (but not d-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of l-nucleotides in the cells, thereby playing a key role in the activity of l-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of l- and d-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog diphosphates.     

Biochemical characterization and crystallization of recombinant 3-phosphoglycerate kinase of Plasmodium falciparum

Human malaria parasite Plasmodium falciparum depends largely on glycolytic pathway for energy metabolism during the intraerythrocytic life stage. Therefore, enzymes of the glycolytic pathway could offer potential drug targets provided novel biochemical and/or structural features of the parasitic enzymes, which distinguish them from the host counterpart, could be identified. 3-Phosphoglycerate kinase (EC 2.7.2.3) catalyzes an important phosphorylation step leading to the production of ATP in the glycolytic pathway. We have expressed recombinant 3-phosphoglycerate kinase of P. falciparum in Escherichia coli. The recombinant protein purified from the soluble fraction of E. coli is enzymatically active. The apparent K(m) values determined for adenosine triphosphate (ATP) and 3-phosphoglycerate (3-PGA) are 0.63 and 0.52 mM, respectively. The enzyme activity was temperature-sensitive. Suramin was found to inhibit the recombinant enzyme with an IC(50) value of 7 microM. We have crystallized the enzyme form in hexagonal space group P6(1)22 (or its enantiomorphic space group) with unit cell parameters a=b=130.7, c=263.9 A. Native data have been collected at 3.0-A resolution.     

Participation of glyceraldehyde-3-phosphate dehydrogenase in the regulation of 2,3-diphosphoglycerate level in erythrocytes

Data are presented concerning the possible participation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in regulation of the glycolytic pathway and the level of 2,3-diphosphoglycerate in erythrocytes. Experimental support has been obtained for the hypothesis according to which a mild oxidation of GAPDH must result in acceleration of glycolysis and in decrease in the level of 2, 3-diphosphoglycerate due to the acyl phosphatase activity of the mildly oxidized enzyme. Incubation of erythrocytes in the presence of 1 mM hydrogen peroxide decreases 2,3-diphosphoglycerate concentration and causes accumulation of 3-phosphoglycerate. It is assumed that the acceleration of glycolysis in the presence of oxidative agents described previously by a number of authors could be attributed to the acyl phosphatase activity of GAPDH. A pH-dependent complexing of GAPDH and 3-phosphoglycerate kinase or 2, 3-diphosphoglycerate mutase is found to determine the fate of 1,3-diphosphoglycerate that serves as a substrate for the synthesis of 2,3-diphosphoglycerate as well as for the 3-phosphoglycerate kinase reaction in glycolysis. A withdrawal of the two-enzyme complexes from the erythrocyte lysates using Sepharose-bound anti-GAPDH antibodies prevents the pH-dependent accumulation of the metabolites. The role of GAPDH in the regulation of glycolysis and the level of 2,3-diphosphoglycerate in erythrocytes is discussed.     

Isolation and characterization of the 'photosynthetic' phosphoglycerate kinase from Beta vulgaris.

1. Phosphoglycerate kinase has been isolated from a photosynthetic plant tissue, Beta vulgaris leaves. The purification procedure is described. 2. The best preparation had no detectable impurity on electrophoresis, and had a specific activity comparable with the same enzyme from other sources. 3. The molecular weight was not distinguishably different from that of the yeast or muscle enzyme, as measured by polyacrylamide-dodecylsulphate electrophoresis. Measurement of aromatic and sulphydryl residues indicated a close similarity with the yeast enzyme. The enzyme appears to have substantially lower isoelectric point than phosphoglycerate kinases from other sources. 4. Kinetic studies indicated that the affinities for the substrates MgATP2- and 3-phosphoglycerate were not significantly different from those of the 'glycolytic' yeast enzyme. There was no evidence that the B. vulgaris enzyme had specific properties making it more suitable for its gluconeogenic rather than glycolytic role.     

Analysis of the inhibitory effect of inorganic phosphate on development of four-cell hamster embryos in vitro

Hamster 4-cell stage embryos were cultured in a protein-free, glucose-free medium to study the nature of developmental inhibition by inorganic phosphate (Pi). In the absence of Pi, between 40 and 55% of embryos were able to develop to the blastocyst stage but addition of Pi to the medium reduced this proportion to 5-20%. The inhibition did not appear to be due to contamination of the Pi salt with heavy metals because EDTA did not relieve the effect. Inhibition by Pi showed no dose-response relationship over the range tested (1-350 microM). In contrast, another divalent anion (sulphate) produced no inhibition of 4-cell embryo development at concentrations as high as 5.6 mM. Embryos were less sensitive to inhibition by Pi after the third cleavage division had occurred, and development of mid or late 8-cell embryos was unaffected by Pi. After exposure to Pi for 1 h, embryos could recover and continue development but longer exposure was detrimental to subsequent development. These results indicate that the inhibitory effect is specific to phosphate ions, is not due to contaminants in the Pi salt, is evoked by very low concentrations of Pi, is stage-specific, and is reversible following brief exposure of embryos to Pi. These effects may be artifacts of the culture milieu, or they may reflect some unknown characteristic of the early cleavage stage hamster embryo.     

Association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of glioma cells

A latex phagocytosis technique was used to prepare relatively pure plasma membranes with inside-out orientation. This method was adapted through a number of modifications in order to evaluate the association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of C6 glial cells. As phosphorylation is strictly coupled with transport in these cells, glycolytic enzymes, especially hexokinase, could metabolize glucose in close vicinity to its transporter. Of the enzymes tested, hexokinase is present in considerable quantities on these membranes (nearly 40% of homogenate specific activity), followed by D-glyceraldehyde-3-phosphate dehydrogenase (10%), pyruvate kinase (8%), and 3-phosphoglycerate kinase (1%). Except for hexokinase, the enzyme pattern presented here is different from that published for other membrane preparations.     

Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition

Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.     

ATP-dependent protein kinase activities in the oral pathogen Streptococcus mutans

ATP-dependent protein kinase activities were detected in both membrane and cytoplasmic fractions from the oral pathogen Streptococcus mutans. Different polypeptides were phosphorylated by endogenous kinase(s) in the two fractions. In membranes, five phosphoproteins were detected with apparent masses of 82, 37, 22, 12, and 10 kilodaltons (KD). In cytoplasm, two major acid-stable phosphoproteins were found. One was identified as HPr of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), while the other had an apparent mass of 61 KD. Both of these proteins were phosphorylated on a seryl residue. Fructose 1,6-bisphosphate stimulated phosphorylation of HPr by the kinase and inhibited phosphorylation of the 61-KD protein. In contrast, fructose 1-phosphate, 2-phosphoglycerate, 3-phosphoglycerate, and dihydroxyacetone phosphate inhibited phosphorylation of HPr and stimulated phosphorylation of the 61-KD protein. Several other glycolytic intermediates as well as inorganic phosphate inhibited phosphorylation of either or both proteins. Preincubation of cytoplasm with PEP prior to incubation with ATP reduced the amount of phospho-(seryl)-HPr formed, but not that of the 61-KD phosphoprotein. The latter protein has not yet been identified but has properties that suggest that it may be the protein kinase itself. These results provide evidence for one or more soluble ATP-dependent protein kinases in S mutans that are regulated by glycolytic intermediates and that may play a role in the modulation of carbohydrate uptake and metabolism in this organism. A model for feedback regulation of sugar transport in S mutans, mediated by an allosterically regulated kinase, is presented.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts.

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [gamma-32P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg2+ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

Effect of superoxide dismutase on the development of preimplantation mouse embryos

The role of superoxide dismutase (SOD) was tested on preimplantation development of mouse embryos in vitro. The presence of SOD in ovarian antral follicles and in oviductal and uterine secretions was also investigated. Zygotes from superovulated ICR female mice were cultured in modified Whittingham's T6 medium supplemented with SOD (0 to 370 U) or EDTA (100 muM) at 37 degrees C under 5% CO(2) in air. Supplementation of SOD (370 U) significantly promoted the development of zygotes to the blastocyst stage (45%) as compared to that of the controls (1.4%). This favorable effect of SOD was comparable to that of EDTA and completely suppressed by anti-SOD antibody. Blastocysts cultured with SOD consisted of 78.2+/-10.4 blastomeres and possessed as many blastomeres as those (81.6+/-9.3) developing in vivo; blastocysts cultured with EDTA had significantly fewer blastomeres (42.6+/-13.7). These findings suggest that SOD protects embryos against oxidative insults and that it can be an effective substitute for EDTA for supporting mouse embryo development in vitro. The SOD activity was detected in 3 different lumina from mouse reproductive organs, and SOD was identified as a cytosolic Cu,Zn-SOD on photochemically stained polyacrylamide gels. Our results suggest that oxidative injury may be responsible for developmental retardation of preimplantation-stage mouse embryos in vitro and that Cu,Zn-SOD may play a crucial role in protecting embryos against oxygen toxicity in vivo as well as in vitro.     

Effect of some glycolytic intermediates and palmitoyl-CoA on alpha-glycerophosphate dehydrogenase in mitochondria isolated from liver of triiodothyronine-treated rats

alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.     

Mouse zygote development in culture medium without protein in the presence of ethylenediaminetetraacetic acid

Development of zygotes from a hybrid-inbred (B6D2F1) and two random-bred (CD1 and CF1) strains of mice were compared after culture in several modifications of a simple, chemically defined medium based on Earle's Balanced Salts Solution. When cultured without the addition of protein or the chelating agent, ethylenediaminetetraacetic acid (EDTA), none of the zygotes reached the blastocyst stage. The addition of EDTA or protein significantly improved embryo development to blastocysts (p less than 0.05). The degree of improvement was dependent upon the strain of the female (85% or 91% for B6D2F1, 56% or 45% for CD1, and 19% or 28% for CF1, respectively). The addition of protein to the media in the presence of EDTA did not further improve embryo development. In all supportive conditions, zygotes from B6D2F1 females developed to blastocysts better than those from CD1 or CF1 females; embryos of the latter strain exhibited the lowest rates of development in vitro. Glycine and alanine (20 microM) partially substituted for EDTA; the decreased hybrid-inbred embryo development to blastocysts (20% and 26%, respectively) obtained in the presence of the amino acids suggested, however, that the stimulatory effect of EDTA on embryo development was other than as a source of fixed nitrogen. The rates of development observed with an alternate chelating agent, citric acid (less than or equal to 20% vs. 83% blastocysts, p less than 0.01), although better than the unsupplemented medium, were significantly less effective than EDTA-supplemented medium (83% blastocysts, p less than 0.01). The results of this study suggest that the protective effect of proteins in culture medium may be more important than their nutritive role.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

P24, a glycogen synthase kinase 3 (GSK 3) inhibitor.

A heat resistant glycogen synthase kinase 3 (GSK 3) binding protein, p24, that inhibits its kinase activity at a low magnesium concentration (in a way similar to that of lithium) was found in microtubules from adult rat brains. This protein associates with GSK 3 in microtubules and corresponds to one previously described in the literature as p25, although it has a relative molecular weight of 23472. p24 is a poor substrate for GSK 3 but it could be phosphorylated by other protein kinases such as cAMP dependent protein kinase and cdk 5. Since p24 could form complexes with GSK 3, it may not only regulate GSK 3 activity but also it might act as an anchoring protein for the kinase.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [γ-³²P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg²⁺ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

The effect of in vitro treatment of bovine embryos with IGF-1 on subsequent development in utero to Day 14 of gestation

Culture of bovine embryos with insulin-like growth factor-1 (IGF-1) can improve development to the blastocyst stage and embryo survival following transfer to heat-stressed, lactating dairy cows. Two experiments were conducted to determine whether IGF-1 could improve embryo survival and development at Day 14 after ovulation. In Experiment 1, non-lactating Holstein cows (n=58) were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced in vitro and cultured with or without 100ng/mL IGF-1. At Day 7 after expected ovulation (Day 0), groups of 7-12 embryos were randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and the presence or absence of an embryonic disc was recorded. Recovered embryos were cultured individually for 24h to determine interferon-tau (IFN-tau) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-tau secretion. In Experiment 2, non-lactating (n=56) and lactating (n=35) Holstein cows were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced as described in Experiment 1. At Day 7 after expected ovulation (Day 0), a single embryo was randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and IFN-tau secretion were determined as in Experiment 1. Recovery rate at Day 14 tended (P=0.1) to be higher for recipients that received IGF-1 treated embryos compared to control embryos (43.2% versus 26.1%, respectively). There was no effect of IGF-1 on embryo length or IFN-tau secretion. In conclusion, results suggest that exposure to IGF-1 through Days 7-8 of development does not enhance capacity of embryos to prevent luteolysis. Results of the single embryo-transfer experiment suggested that IGF-1 treatment might affect embryo survival post-transfer as early as Day 14 after ovulation. Further experimentation is warranted to verify this finding.     

Fasciola hepatica: effect of 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide on glycolysis in vitro

In vitro, 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide, a potent fasciolicide, causes a potent concentration-dependent inhibition of glucose uptake by mature Fasciola hepatica. In F. hepatica treated with the disulfonamide and then fed [U-¹⁴C]glucose, there was a 60% inhibition of glucose utilization and a corresponding inhibition of acetate and propionate formation. Treated fluke parasites possessed much lower levels of adenosine triphosphate, phosphoenolpyruvate, glucose 6-phosphate, and fructose 6-phosphate than untreated parasites and contained higher levels of glycerol and the free sugars fructose and mannose. Direct measurement of the effect of the disulfonamide on the glycolytic enzymes of F. hepatica demonstrated that 3-phosphoglycerate kinase (EC 2.7.2.3) and phosphoglyceromutase (EC 2.7.5.3) were inhibited. It is therefore suggested that the fasciolicidal activity of 4-amino-6-trichloroethenyl-1, 3-benzenedisulfonamide is due to inhibition of the enzymes 3-phosphoglycerate kinase and phosphoglyceromutase which effectively blocks the Embden-Myerhof glycolytic pathway.