Reevaluation of the "glycolytic complex" in muscle: a multitechnique approach using trout white muscle

Preliminary characterization of the "glycolytic complex," formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a pKa of 7.3 in 30 mM imidazole. PFK binding increased at lower pH values; at 150 mM KCl the apparent pKa value is 6.5. Experiments with polyethylene glycol 8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium--with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F.D. Shaw, and D.J. Morton (1980) Biochem. J. 186, 105-109). In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in vivo: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (10-30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies.     

Glyceraldehyde-3-phosphate dehydrogenase,a glycolytic enzyme present in the periplasm of Aeromonas hydrophila

This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10–20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A₆₀₀ = 1.1–1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Continuous ethanol production by a flocculating strain of Kluyveromyces marxianus: bioreactor performance

A flocculating strain of Kluyveromyces marxianus was used for alcoholic fermentation in a continuous bioreactor working with zero residual concentration in effluent. Specific kinetic parameters were improved by increasing dilution rate, which is similar to results obtained with ultrafiltration systems. Specific biomass accumulation rate had always a value greater than 92.5% of specific biomass growth rate and was independent of the dilution rate. Productivity is shown to be 12.5 times greater than in conventional continuous operation and is directly proportional to dilution rate. Maximum biomass concentration also presents a linear relationship with dilution rate. The largest obtained biomass concentration is 8 times greater than in a conventional continuous fermentor.     

GroEL and protein disulfide isomerase each binds with folding intermediates of D-glyceraldehyde-3-phosphate dehydrogenase released from complexes formed with the other

Simultaneous presence of two chaperones, GroEL and protein disulfide isomerase (PDI), assists the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an additive way. Delayed addition of chaperones to the refolding solution after dilution of denatured GAPDH indicates an interaction with intermediates formed mainly in the first 5 min for PDI and formed within a longer time period for GroEL-ATP. The above indicate that the two chaperones interact with different folding intermediates of GAPDH. After delayed addition of one chaperone to the refolding mixture containing the other at 4°C, GroEL binds with all GAPDH intermediates dissociated from PDI, and PDI interacts with the intermediates released from GroEL during the first 10–20 min. It is suggested that the GAPDH folding intermediates released from the chaperone-bound complex are still partially folded so as to be rebound by the other chaperone. The above results clearly support the network model of GroEL and PDI.     

Variation and modeling of the probability of plasmid loss as a function of growth rate of plasmid-bearing cells of Escherichia coli during continuous cultures

A large number of models concerning cultures of genetically engineered bacteria have been described. Among them, some are specifically adapted to continuous cultures and lead to the determination of two variables: (i) the difference in the specific growth rates between plasmid-carrying cell and plasmid-free cells (deltamu) and (ii) the frequency of plasmid loss by plasmid-containing cells (p(r)mu(+)). Until now, studies have been performed on the global expression p(r)mu(+) and deltamu, whose value during continuous assays have been supposed approximately constant (mean value) and not on separate values of both terms p(r) and mu(+), respectively, probability of plasmid loss and specific growth rate of the plasmid-carrying cells. So far these studies do not allow examination of the relationship between these two last parameters. Experimental results were obtained with Escherichia coli C600 galk (GAPDH), a genetically engineered strain that synthetizes an elevated quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). From data obtained during continuous cultures, it is shown that during an assay, deltamu, and p(r)mu(+) do not remain constant. An appropriate mathematical analysis of the expression of mu(-) (specific growth rate of the plasmid-free cells) and mu(+) has been built up. This allows the evaluation of the values of mu(+) and mu(-) during the continuous cultures carried out at different dilution rates. Values of p(r) have been calculated from these data. Indeed our results show that p(r) increases with mu(+). A modeling approach which allows correct simulation of this variation is also proposed. This model is derived from the Hill equation regarding cooperative binding of enzymic type reaction. (c) 1993 John Wiley & Sons, Inc.     

Participation of chaperonin GroEL in the folding of D-glyceraldehyde-3-phosphate dehydrogenase. An approach based on the use of different oligomeric forms of the enzyme immobilized on sepharose

The binding of denatured B. stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the E. coli chaperonin GroEL was investigated in two systems: (1) GroEL immobilized on Sepharose via a single subunit was titrated with urea-denatured soluble GAPDH and (2) a Sepharose-bound denatured GAPDH monomer was titrated with soluble GroEL. Similar apparent K _D values for the complex GroEL·GAPDH were obtained in both cases (0.04 and 0.03 M, respectively), the stoichiometry being 1.0 mol chaperonin per GAPDH subunit in the system with the immobilized GroEL and 0.2 mol chaperonin per Sepharose-bound GAPDH monomer. Addition of GroEL and Mg·ATP to a reactivation mixture increased the yield of reactivation of both E. coli and B. stearothermophilus GAPDHs. Incubation of the Sepharose-bound catalytically active tetrameric and dimeric GAPDH forms with the protein fraction of a wild-type E. coli cell extract resulted in the binding of GroEL to the dimer and no interaction with the tetrameric form. These data suggest that GroEL may be capable of interacting with the interdimeric contact regions of the folded GAPDH dimers.     

Response of a continuous anaerobic culture to periodic variation of the feeding mash concentration

The authors studied the influence of periodic variations (one cycle per 24 hr) of the feeding mash concentration of a continuous anaerobic culture of Saccharomyces cerevisiae in sugarcane molasses media at constant dilution rate. It was observed that the average yield coefficients during the transient state were practically equal to the yield coefficient obtained during steady-state experiments. It was also observed, in each experiment, that the average specific growth rate during the transient state was equal to the dilution rate.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote,Trichomonas vaginalis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD⁺ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica—Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium. Correspondence to: M. Müller     

Study of the Properties of Phosphorylating D-Glyceraldehyde-3-phosphate Dehydrogenase

The properties of the active center of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are considered with emphasis on the structure of anion-binding sites and their role in catalysis. The results of studies on the molecular mechanism of the effect of NAD⁺ on the enzyme conformation are discussed. Experimental evidence is presented supporting the idea that negative cooperativity of NAD⁺ binding and half-of-the-sites reactivity exhibited by GAPDH are generated by different mechanisms. Data obtained with rabbit muscle and Escherichia coli GAPDH point to preexisting asymmetry in these tetramers. Structural determinants that can control the transition of the tetramer from the symmetric to the asymmetric state were found.     

Glucose and acetate influences on the behavior of the recombinant strainEscherichia coli HB 101 (GAPDH)

Summary This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) byE. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L^–1 of biomass and 6 g L^–1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L^–1, and in fed-batch experiments for glucose availability of 10 g h^–1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.     

Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.     

Kinetic behavior of mixed populations of activated sludge

The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single-stage isothermal continuous flow completely mixed aeration tank. A series of experiments were carried out at various dilution rates using glucose as the growth limiting substrate. The steady-state behavior of the system was observed at each dilution rate and the results were found to fit fairly well with the steady-state equation bayed on the Monod model with an endogenous respiration term included, i.e., μ = μ_mS/(K_s + S) ? K_d. The growth kinetics of cells harvested at steady state for each dilution rate were studied using batch experiments. The multiple response data of the system as functions of time were used to estimate the parameter values in the above kinetic model. It was found that values of the growth parameters changed significantly and systematically with cell population. For example, values of μ_m were high at high dilution rates and low at low dilution rates. It was also found that only those batch growth parameters from cells obtained at fairly high dilution rates are comparable with those estimated by the results of steady-state operations. The results of this investigation suggest that (1) different cell populations pre dominated at different steady-state dilution rates, with high dilution rates resulting in predominantly fast-growing organisms and low dilution rates resulting in predominantly slow-growing cells, and (2) risk exists in any randomly picked batch experiment to predict the steady-state behavior of the system when heterogeneous microbial populations must be used.     

Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants

The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction (PCR) cycle in the stroma. Glyceraldehyde-3-phosphate dehydrogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enzymes which catalyse this reversible conversion. We report on the engineering of an antisense RNA construct directed against the tobacco (Nicotiana tabacum L.) chloroplastlocated GAPDH (A subunit). The construct was integrated into the tobacco genome by Agrobacterium-mediated transformation of leaf discs. Of the resulting transformants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% of wild-type (WT) activities. Segregation analysis of the kanamycin-resistance character in self-pollinated T₁ seed from each of the five transformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert. T₁ progeny from GAP-R was used to generate plants with GAPDH activities ranging from WT levels to around 7% of WT levels. These were used to study the effect of variable GAPDH activities on metabolite pools for ribulose1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO₂ assimilation and other gasexchange parameters. The RuBP pool size was linearly related to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO₂ assimilation was not affected until RuBP levels dropped to 30–40% of WT levels. That is, the CO₂ assimilation rate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol·(mol site)^–1 while the ratio for WT plants was 4–5 mol·m(mol site)^–1. Leaf conductance was not reduced in leaves with reduced GAPDH activities, resulting in an increase in the ratio of intercellular to ambient CO₂ partial pressure. Conductance in plants with reduced GAPDH activities was still sensitive to CO₂ and showed a normal decline with increases in CO₂ partial pressure. Although PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP. Concomitant gas-ex change and chlorophyll a fluorescence measurements indicated that photosynthesis changed from being Rubisco-limited to being RuBP-regeneration-limited at a lower CO₂ partial pressure in the antisense plants than in WT plants. Photosynthetic electron transport was down-regulated by the build-up of a large proton gradient and the electron-transport chain did not become over-reduced due to a shortage of NADP. Plants with severely reduced GAPDH activity were not photoinhibited despite the continuous presence of a large thylakoid proton gradient in the light. Along with plant size, Rubisco activity, leaf soluble protein and chlorophyll content were reduced in plants with the lowest GAPDH activities. We conclude that chloroplastic GAPDH activity does not appear to limit steady-state photosynthetic CO₂ assimilation at ambient CO₂. This is because WT leaves maintain the ratio of RuBP per Rubisco site about twofold higher than the level required to achieve a maximal rate of CO₂ assimilation.Abbreviations and Symbols bp base pairs - DHAP dihydroxy-acetone phosphate - GAPDH glyceraldehyde-3-phosphate dehy-drogenase - PCR photosynthetic carbon reduction - PGA 3-phosphoglycerate - p_i intercellular CO₂ partial pressure - q_NP non-photochemical fluorescence quenching - q_Q photochemicalfluorescence quenching - PSII quantum efficiency of electronflow through PSII - Rubisco ribulose-1,5-bisphosphate carboxy-lase-oxygenase - RuBP ribulose-1,5-bisphosphate - WT wild type We thank Karin Harrison, Prue Kell, Anne Gallagher and Barbara Setchell for excellent technical assistance. G.D.P. and S.V.C. acknowledge support from QE II Research Fellowships (Australian Research Council).     

Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-β-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of α subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.