Presence Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Expression of glyceraldehyde-3-phosphate dehydrogenase on the surface of Clostridium perfringens cells

During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C. perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin. Although GAPDH has no signal sequence, it is expressed on the cell surface of many microorganisms. The presence of GAPDH on the surface of C. perfringens cells was analyzed using ELISA and flow cytometry analyses; purified rGAPDH bound to the surface of C. perfringens cells. As autolysin is reportedly involved in the binding of GAPDH to the cell surface, we evaluated the interaction between rGAPDH and the C. perfringens autolysin Acp by both ELISA and ligand blotting assay. These assays revealed that rGAPDH binds to the catalytic domain of Acp but not the cell wall binding domains. These results suggest that autolysin mediates expression of GAPDH on the surface of C. perfringens cells and indicate a possible moonlighting function for GAPDH in binding both Fn and plasminogen.

Cryptic Disorder Out of Disorder: Encounter between Conditionally Disordered CP12 and Glyceraldehyde-3-Phosphate Dehydrogenase

Among intrinsically disordered proteins, conditionally disordered proteins undergo dramatic structural disorder rearrangements upon environmental changes and/or post-translational modifications that directly modulate their function. Quantifying the dynamics of these fluctuating proteins is extremely challenging but paramount to understanding the regulation of their function. The chloroplast protein CP12 is a model of such proteins and acts as a redox switch by formation/disruption of its two disulfide bridges. It regulates the Calvin cycle by forming, in oxidized conditions, a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and then phosphoribulokinase. In this complex, both enzymes are inactive. The highly dynamic nature of CP12 has so far hindered structural characterization explaining its mode of action. Thanks to a synergistic combination of small-angle X-ray scattering, nuclear magnetic resonance and circular dichroism that drove the molecular modeling of structural ensembles, we deciphered the structural behavior of Chlamydomonas reinhardtii oxidized CP12 alone and in the presence of GAPDH. Contrary to sequence-based structural predictions, the N-terminal region is unstable, oscillates at the ms timescale between helical and random conformations, and is connected through a disordered linker to its C-terminus, which forms a stable helical turn. Upon binding to GAPDH, oxidized CP12 undergoes an induced unfolding of its N-terminus. This phenomenon called cryptic disorder contributes to decrease the entropy cost and explains CP12 unusual high affinity for its partners.

Improvement of expression level of polysaccharide lyases with new tag GAPDH in E. coli

Escherichia coli (E. coli) is widely used to express a variety of heterologous proteins. Efforts have been made to enhance the expression level of the desired protein. However, problems still exist to regulate the level of protein expression and therefore, new strategies are needed to overcome those issues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is properly expressed in E. coli might play a leading role and increase the expression levels of the target proteins. In this study, GAPDH was fused with a target enzyme, ChSase ABC I, an endoeliminase and polysaceharide lyase. Our results confirmed this hypothesis and indicated that GAPDH boosted the expression level of ChSase ABC I with an increase of 2.25 times, while the enzymatic activity with an increase of 2.99 times. The hypothesis were also supported by RT-PCR study and GAPDH was more effective in enhancing the expression level and enzymatic activity as compared to MBP, which is commonly used as fused tag and can improve the soluble expression of target protein. addition, the expression level and enzymatic activity of other polysaceharide lyases were also improved in the presence of GAPDH. The findings of this study prove that GAPDH has a strong effect on enhancing the expression level and enzymatic activity of the target proteins.

The activation of glycolysis performed by the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the model system

Influence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) on glycolysis was investigated. The addition of GAPN—which oxidizes glyceraldehyde-3-phosphate directly to the 3-phosphoglyceric acid—led to the strong increase in the rate of lactate accumulation in the rat muscle extract with low ADP content. The lactate accumulation was also observed in the presence of GAPN in rat muscle extract, which contained only ATP and no ADP. This can be the evidence of the “futile cycle” stimulated by GAPN. Here ADP can be regenerated from ATP by the phosphoglycerate kinase reaction. The high resistance of GAPN from Streptococcus mutans towards inactivation by natural oxidant—H 2O 2 was showed. This feature distinguishes GAPN from phosphorylating glyceraldehyde-3-phosphate dehydrogenase, which is very sensitive to modification by hydrogen peroxide. A possible role of the oxidants and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the regulation of glycolysis is discussed.

An improved assay for UDPglucose pyrophosphorylase and other enzymes that have nucleotide products.

UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose 1-phosphate plus UTP. Several assay methods for this enzyme have been described but the only one that can be used to investigate the specificity with respect to various UDPsugars is based on coupling to UTP formation. This assay employs phosphoglycerate kinase to catalyse the formation 1,3- bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phosphate dehydrogenase. We have found that the activity of phosphoglycerate kinase towards UTP is low which limits the usefulness of the assay to very low rates, in agreement with the published recommendation of Hansen et al. Here it is shown that the dynamic range of the assay is increased by more than five fold on addition of nucleoside diphosphate kinase and ADP, which convert UTP to the preferred phosphoglycerate kinase substrate, ATP. It is also shown that the improved assay is suitable for enzymes with other nucleotide triphosphate products.

Glycolytic enzymes and assembly of microtubule networks

The ability of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase (PK), and lactate dehydrogenase muscle-type (LDH m), to generate interactive microtubule networks was investigated. Bundles have previously been defined as the parallel alignment of several microtubules and are one form of microtubule networks. Utilizing transmission electron microscopy, interactive networks of microtubules as well as bundles were readily observed in the presence of GAPDH, aldolase, or PK. These networks appear morphologically as cross-linked microtubules, oriented in many different ways. Light scattering indicated that the muscle forms of GAPDH, aldolase, PK, and LDH m caused formation of the microtubule networks. Triose phosphate isomerase (TPI) and lactate dehydrogenase heart-type (LDH H), glycolytic enzymes which do not interact with tubulin or microtubules, did not produce bundles, or interactive networks. Sedimentation experiments confirmed that the enzymes that cross-link also co-pellet with the microtubules. Such cross-linking of microtubules indicate that the enzymes are multivalent with the capability of simultaneous binding to more than one microtubule.

Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads

Isolated triadic proteins were employed to investigate the molecular architecture of the triad junction in skeletal muscle. Immunoaffinity-purified junctional foot protein (JFP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase and partially purified dihydropyridine (DHP) receptor were employed to probe protein-protein interactions using affinity chromatography, protein overlay and crosslinking techniques. The JFP, an integral protein of the sarcoplasmic reticulum (SR) preferentially binds to GAPDH and aldolase, peripheral proteins of the transverse (T)-tubule. No direct binding of JFP to the DHP receptor was detected. The interactions of JFP with GAPDH and aldolase appear to be specific since other glycolytic enzymes associated with membranes do not bind to the JFP. The DHP receptor, an integral protein of the T-tubule, also binds GAPDH and aldolase. A ternary complex between the JFP and the DHP receptor can be formed in the presence of GAPDH. In addition, the DHP receptor binds to a previously undetected Mr 95 K protein which is distinct from the SR Ca2+ pump and phosphorylase b. The Mr 95 K protein is an integral protein of the junctional domain of the SR terminal cisternae. It is also present in the newly identified "strong triads" (accompanying paper). From these findings, we propose a new model for the triad junction.

A simple modification to an enzymic method for the preparation of[ γ- 32P]ATP free of salt and buffer

An existing enzymic method for preparing [ γ- 32P]ATP from 32 P i has been modified toyield [ γ- 32P]ATP free of salt and buffer. 32P is incorporated into the γ-position of ATP by isotopic exchange in the presence of glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase. Unreacted 32 P i is separated from [ γ- 32P]ATP by column chromatography on Dowex 1 bicarbonate. [ γ- 32P]ATP is eluted with 2 m triethylammonium bicarbonate, which is then completely removed by freeze-drying.