Glyceraldehyde-3-phosphate Dehydrogenase Binding Research Articles

Neuroprotection by deprenyl and other propargylamines: glyceraldehyde-3-phosphate dehydrogenase rather than monoamine oxidase B.

Deprenyl and other propargylamines are clinically beneficial in Parkinson's disease (PD). The benefits were thought to depend on monoamine oxidase B (MAO-B) inhibition. A large body of research has now shown that the propargylamines increase neuronal survival independently of MAO-B inhibition by interfering with apoptosis signaling pathways. The propargylamines bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GAPDH binding is associated with decreased synthesis of pro-apoptotic proteins like BAX, c-JUN and GAPDH but increased synthesis of anti-apoptotic proteins like BCL-2, Cu-Zn superoxide dismutase and heat shock protein 70. Anti-apoptotic propargylamines that do not inhibit MAO-B are now in PD clinical trial.

The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase

The cytoplasmic domain of band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and band 4.1 to band 3: (1) the predominant or even sole band 3 oligomer forming the binding site is the tetramer. (2) The band 3 tetramer can bind up to four tetramers of GAPDH. (3) The band 3/GAPDH complexes are unstable. (4) Artificially stabilized band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the band 3 tetramer, and that ternary complexes of band 3 tetramers, GAPDH and aldolase do exist.

Binding of estrogen and progesterone-BSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDH enzyme activity: physiological implications

In this study rat brain solubilized plasmalemma-microsomal fractions (B-P3) or cytosolic fractions were applied to P-3-BSA (progesterone linked to BSA at C-3 position) and E-6-BSA (17β-estradiol linked to BSA at C-6 position) affinity columns. It is interesting that a 37 kDa protein was retained by both columns which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing. The 37 kDa protein (GAPDH) was not retained by either a control BSA conjugated affinity column or a corticosterone-BSA affinity column. E-6-BSA bound to GAPDH with higher binding affinity than P-3-BSA or T-3-BSA (testosterone linked to BSA at C-3 position) affinity columns. In addition, the binding of 17β-E-6-BSA to GAPDH was impeded by free estrogen (17β-estradiol) completely. Binding studies of E-6-BSA and P-3-BSA to commercial GAPDH from rabbit skeletal muscle using radiolabeled ligand binding assays revealed that P-3-BSA had 10× lower GAPDH binding affinity than E-6-BSA. Next, the effects of estrogen and progesterone on GAPDH activity were studied. Rapid and significant increases in V max and changes in K m were observed by the addition of 10 nM estradiol, whereas 100 nM progesterone decreased only V max significantly. Testosterone, corticosterone, 17α-estradiol, and diethylstilbestrol did not affect the enzyme activity. The results indicate that GAPDH is a target site for 17β-estradiol and progesterone and suggest possible roles in the regulation of cellular metabolism and synaptic remodeling in which GAPDH has been reported to be involved.

Functional significance of the interaction of hepatitis A virus RNA with glyceraldehyde 3-phosphate dehydrogenase (GAPDH): opposing effects of GAPDH and polypyrimidine tract binding protein on internal ribosome entry site function.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a cellular enzyme involved in glycolysis, binds specifically to several viral RNAs, but the functional significance of this interaction is uncertain. Both GAPDH and polypyrimidine tract binding protein (PTB) bind to overlapping sites in stem-loop IIIa of the internal ribosome entry site (IRES) of Hepatitis A virus (HAV), a picornavirus. Since the binding of GAPDH destabilizes the RNA secondary structure, we reasoned that GAPDH may suppress the ability of the IRES to direct cap-independent translation, making its effects antagonistic to the translation-enhancing activity of PTB (D. E. Schultz, C. C. Hardin, and S. M. Lemon, J. Biol. Chem. 271:14134-14142, 1996). To test this hypothesis, we constructed plasmids containing a dicistronic transcriptional unit in which the HAV IRES was placed between an upstream GAPDH-coding sequence and a downstream Renilla luciferase (RLuc) sequence. Transfection with this plasmid results in overexpression of GAPDH and in RLuc production as a measure of IRES activity. RLuc activity was compared with that from a control, null-expression plasmid that was identical except for a frameshift mutation within the 5' GAPDH coding sequence. In transfection experiments, GAPDH overexpression significantly suppressed HAV IRES activity in BSC-1 and FRhK-4 cells but not in Huh-7 cells, which have a significantly greater cytoplasmic abundance of PTB. GAPDH suppression of HAV translation was greater with the wild-type HAV IRES than with the IRES from a cell culture-adapted virus (HM175/P16) that has reproducibly higher basal translational activity in BSC-1 cells. Stem-loop IIIa RNA from the latter IRES had significantly lower affinity for GAPDH in filter binding experiments. Thus, the binding of GAPDH to the IRES of HAV suppresses cap-independent viral translation in vivo in African green monkey kidney cells. The enhanced replication capacity of cell culture-adapted HAV in such cells may be due in part to reduced affinity of the viral IRES for GAPDH.

Specific Interaction between the Hepatitis Delta Virus RNA and Glyceraldehyde 3-Phosphate Dehydrogenase: An Enhancement on Ribozyme Catalysis

Replication of hepatitis delta virus (HDV) RNA occurs in the nuclei of infected cells. The replication is mediated by cellular factors containing an RNA polymerase II-like enzyme activity through a double rolling-circle mechanism and is regulated by delta antigens. In this study, UV cross-linking experiments were carried out to examine interactions between HDV RNA and proteins present in HeLa nuclear extract. Cellular proteins with molecular mass of 23 (p23), 36 (p36), 38 (p38), and 58 (p58) kDa bound to full-length HDV RNA of both genomic and antigenomic strands. Deletion analysis on the antigenomic strand mapped the interacting domain within a 79-nucleotide fragment but not at the ends of the rod-shaped viral RNA structure. The specificity of the RNA-protein interactions was demonstrated by competition experiments and the specific HDV RNA-binding proteins were purified through column chromatography. Electrophoresis mobility shift assay with the purified fractions demonstrated that the interaction between p36 and HDV RNA was relatively stable even in the presence of 0.5 M NaCl. Biochemical analysis including protein microsequencing identified the p36 as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RNase footprinting indicated that the UC-rich domain between nucleotides 379 and 414 of the HDV antigenomic RNA was involved in the GAPDH binding. Functional studies further demonstrated an enhancing effect of GAPDH on the ribozyme activity of HDV antigenomic RNA. In addition, in the presence of HDV RNA cellular GAPDH relocalized from the cytoplasm to the nucleus where HDV replication occurs. These results suggest that GAPDH is involved in the replication of HDV.

Functional Significance of the Interaction of Hepatitis A Virus RNA with Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH): Opposing Effects of GAPDH and Polypyrimidine Tract Binding Protein on Internal Ribosome Entry Site Function

Functional Significance of the Interaction of Hepatitis A Virus RNA with Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH): Opposing Effects of GAPDH and Polypyrimidine Tract Binding Protein on Internal Ribosome Entry Site Function

Conformational structure and binding mode of glyceraldehyde-3-phosphate dehydrogenase to tRNA studied by Raman and CD spectroscopy

Recently it has been suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a role in nuclear tRNA export. As the structural basis of binding of GAPDH to tRNA is as yet unknown, we have employed Raman and CD spectroscopy as probes of the solution structures of GAPDH from rabbit and tRNA Phe from brewers yeast. Additionally, we have obtained the Raman and CD spectra of GAPDH when bound to tRNA Phe. In the complex we find the following results: (a) The most part of the tRNA Phe structure is conserved, but with a slight perturbation toward a B-like form. (b) No significant changes in the secondary structure of the protein upon binding are observed. (c) The surface enhanced Raman spectra are consistent with a GAPDH-tRNA Phe complex molecular model that involves the insertion of tRNA Phe into the GAPDH tetramer groove containing the R and P axes. (d) The specific interactions that occur between GAPDH and the tRNA Phe involve, mainly, stacking between nucleobases and aromatic amino-acid residues, and ionic interactions of basic amino-acid residues with phosphate groups of the ribose-phosphate backbone. The above stacking interactions are also supported by the significant relatedness that we have found between an amino-acid sequence (residues 303–308) of GAPDH and RNP2 binding motifs of some RNA binding proteins.

Cotransport of glyceraldehyde-3-phosphate dehydrogenase and actin in axons of chicken motoneurons.

1. To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [35S]methionine and, 1-20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I-Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography. 2. In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 3. The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods. 4. Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3-5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity. 5. Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.

Membrane-bound glyceraldehyde-3-phosphate dehydrogenase and multiphasic erythrocyte sugar transport.

Net sugar import by human erythrocytes consists of ATP-modulated rapid and slow phases while sugar export consists of a single slow phase. We have proposed that this behaviour results from obligate substrate tunnelling from transporter to bulk cytosol through a complex containing high-affinity, low-capacity sugar binding sites (Cloherty, Sultzman, Zottola & Carruthers, 1995). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to compartmentalize ATP delivery to erythrocyte membrane ATPases and interact directly with the erythrocyte glucose transporter in vitro. The present study examines the possibility that GAPDH is an obligate component of the hypothesized sugar-binding complex. GAPDH remains associated with the erythrocyte membrane following cell lysis and remains associated with the cytoskeleton under conditions where more than 99% of the membrane glucose transport protein (GLUT1) is released by detergent (Triton X-100). GAPDH is released from erythrocyte membranes upon exposure to Mg.ATP or to NADH. ATP displacement of membrane-bound GAPDH is half-maximal at 200 microM ATP and appears to involve ATP interaction with multiple, co-operative sites. GAPDH interaction with purified tetrameric GLUT1 is saturable, co-operative and also inhibited by ATP. ATP inhibition of GAPDH binding to purified tetrameric GLUT1 is less effective than ATP inhibition of GAPDH binding to intact erythrocyte membranes. Removal of cellular GAPDH by exposing erythrocyte membranes to NADH prior to membrane resealing neither affects ATP modulation of sugar transport nor reduces biphasic net sugar uptake to a single phase. We conclude that ATP-sensitive GAPDH interaction with the cytoplasmic surface of erythrocyte membranes and GLUT1 is responsible neither for ATP modulation of sugar transport nor for multiphasic net sugar import by human red cells.

S-Nitrosylation of Glyceraldehyde-3-Phosphate Dehydrogenase Decreases the Enzyme Affinity to the Erythrocyte Membrane

The effects of nitric oxide (NO) or related molecules on the binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the red blood cell (RBC) membrane were investigated. It was demonstrated that submillimolar concentrations of the NO donor sodium nitroprusside (SNP) not only strongly inactivated GAPDH by S-nitrosylation of the enzyme thiols but also decreased the binding affinity of GAPDH for the RBC membrane. In fact, the incubation with SNP for 60 min at 30°C and at a concentration >50 μM induced the dissociation of the native GAPDH from the white unsealed membranes (standard ghosts) in a concentration-dependent manner with a partial recovery of the enzyme activity and thiols when SNP concentrations higher of 1 mM were used. Binding experiments under saturating conditions indicate aKavalue for the nitrosylated GAPDH of 3.5 ± 0.8 × 106M−1, which was more than 50% less than theKavalue of 7.6 ± 0.6 × 106M−1observed for the native enzyme. These data were also confirmed in reassociation experiments under nonsaturating conditions. Dithiothreitol (DTT), which at concentrations of less than 1 mM catalyzed the S-nitrosylation of GAPDH and the consequent modification of the binding properties described above, at concentrations higher than 5 mM restored both the enzyme activity and the binding properties. Furthermore, the enzyme–membrane association induced before the incubation step afforded at least partial protection from the loss of titrable thiols and from the inactivation induced either spontaneously or by SNP. Taken together, these data not only confirm the key role of the active site cysteine residues in the catalytic function of GAPDH but also suggest that they may be involved in the NO-dependent regulation of GAPDH binding to the RBC membrane.

Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.

In this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (k[on] =2.8 X 10(4) M(-1) X s[-1]) and dissociated extremely slowly (k[off]=5.9 X 10(-5) s[-1]), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2 microM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.

Glycolytic enzyme binding in Otala lactea hepatopancreas: effect of taxol, colchicine and cytochalasin B and D on the in vivo enzyme distribution.

The effect of cytoskeleton modulators on glycolytic enzyme binding was examined in the hepatopancreas of Otala lactea in an attempt to identify potential cellular binding sites. Binding was followed by measuring phosphofructokinase (PFK), aldolase (ALD), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) distribution between low speed pellets (12,000 xg), high speed pellets (100,000 xg) and high speed supernatants. Taxol (which stabilizes microtubules), colchicine (which destabilizes microtubules) and cytochalasin B and D (which destabilize F-actin filaments) were added to the homogenate prior to centrifugation. Addition of taxol increased the amount of PFK associated with the high speed pellet. Cytochalasin B and D reduced the binding of PFK and PK to the low speed pellet. ALD and GAPDH binding were unaffected by any treatment. Lowering the pH of the crude homogenate increased PFK binding to the low speed pellet by 33%. This effect could be reversed by addition of cytochalasin B and D suggesting that pH influences the PFK-F-actin interaction in vivo. The differential binding response of PFK, PK, ALD and GAPDH to added effectors suggests that, in the cell, PFK and PK are bound to different subcellular structural elements than are ALD and GAPDH.

Enhancement of Hammerhead Ribozyme Catalysis by Glyceraldehyde-3-phosphate Dehydrogenase

A specific tumour necrosis factor alpha ribozyme (TNF-α-Rz) binding activity has been purified and identified by N-terminal microsequencing as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The purified protein as well as commercial GAPDH binds tightly to TNF-α ribozyme compared to a variety of other ribozymes and RNAs. Binding of GAPDH to the TNF-α-Rz and its derivatives was inhibited by NAD+ and ATP, suggesting that the GAPDH Rossmann fold structure is a part of the ribozyme binding site. Interestingly, GAPDH increased the in vitrocleavage rates of hammerhead ribozymes by up to 25-fold, while no significant stimulation was observed with the lactate dehydrogenase (LDH). This effect was found to be due to the unfolding activity of GAPDH. In fact, pulse – chase experiments demonstrate directly that GAPDH has the capacity to accelerate the ribozyme/substrate association, especially of ribozymes and/or substrates whose predicted secondary structure might interfere with the association step. Under our conditions, the presumed unfolding activity of GAPDH also enhances the turnover of ribozymes by increasing the rate of product dissociation, although only for short cleavage products. Longer duplexes required more incubation time to dissociate. In vitronon-specific interaction of the GAPDH with hammerhead ribozymes and RNA substrates was found to be adequate for the cleavage enhancement effect to occur. However, an analysis of the ability of various prototypical ribozymes to inhibit the expression of interleukin-2 suggests that the addition of a sequence having a high affinity for GAPDH improves the efficacy of ribozymes in the cells. Thus, the characterization of cellular proteins with unfolding activity, which specifically bind to hammerhead ribozyme, should facilitate the design of a more effective ribozyme in vivo.

Glyceraldehyde-3-phosphate Dehydrogenase Selectively Binds AU-rich RNA in the NAD+-binding Region (Rossmann Fold)

A 36-kDa protein that binds AU-rich RNA was purified from human spleen and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH has been previously demonstrated to bind tRNA with high affinity. Competition studies suggested that cytoplasmic GAPDH binds the AU-rich elements (AREs) of lymphokine mRNA 3'-untranslated regions with higher affinity than tRNA. The AUUUA-specific RNA binding activity of GAPDH was inhibited by NAD+, NADH, and ATP in a concentration-dependent manner, suggesting that RNA binding of GAPDH might involve the NAD(+)-binding region, or dinucleotide-binding (Rossmann) fold. This hypothesis was supported by experiments that localized RNA binding to the predicted N-terminal 6.8-kDa peptide, known to be involved in the formation of the NAD(+)-binding domain. The direct demonstration of ARE-specific binding protein activity localized to the NAD(+)-binding region of GAPDH supports the general concept that enzymes containing this domain may exhibit specific RNA binding activity and play additional roles in nucleic acid metabolism. Finally, cytoplasmic GAPDH was found in the polysomal fraction of T lymphocytes. Thus, the RNA binding specificity of GAPDH as well as its localization within the cell merit its strong consideration as a protein important in the regulation of ARE-dependent mRNA turnover and translation in addition to its well described role in glycolysis.

Rat Brain Glyceraldehyde‐3‐Phosphate Dehydrogenase Interacts with the Recombinant Cytoplasmic Domain of Alzheimer's β‐Amyloid Precursor Protein

Abundant senile plaques are a histological hallmark in the brain of Alzheimer's disease patients. Such plaques consist of, among many other constituents, aggregated beta A4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimer's disease. To study possible interactions of brain proteins with beta A4 amyloid or other fragments of APP, beta A4 amyloid and beta A4 amyloid extended to the C-terminus of APP were recombinantly produced as fusion proteins termed "Amy" and "AmyC," respectively. Using Amy and AmyC affinity chromatography, a 35-kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C-terminus of APP. This 35-kDa protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the Vmax of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.

Rotational diffusion of band 3 in erythrocyte membranes. 2. Binding of cytoplasmic enzymes

Time-resolved phosphorescence anisotropy has been used to study the rotational diffusion of eosin-labeled human erythrocyte band 3 in the presence of an enzyme bound at its cytoplasmic pole. With increasing amounts of G3PD (glyceraldehyde-3-phosphate dehydrogenase) added to ghosts, the infinite time anisotropy (r infinity) increases, and at saturating concentrations, very little decay of the anisotropy r(t) occurs at all. These phenomena are reversed by elution of the enzyme with 150 mM NaCl. Prior proteolytic removal of the N-terminal 41-kDa cytoplasmic fragment of band 3 also disenables the G3PD effect. When ghosts are stripped of their residually bound G3PD, a small reduction in the fraction of immobile band 3 is observed. Finally, titration of band 3 sites with aldolase shows effects on the r(t) qualitatively similar to those observed with G3PD. On the basis of our interpretation of the heterogenous anisotropy decay of eosin-labeled band 3 [Matayoshi, E. D., & Jovin, T. M. (1991) Biochemistry (preceding paper in this issue)], we conclude that the binding of G3PD and aldolase results in the immobilization of band 3 oligomers.

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 p K a of 7.3 in 30 m m imidazole. PFK binding increased at lower pH values; at 150 m m KC1 the apparent p K a 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.

Subcellular enzyme binding in glycolytic control: in vivo studies with fish muscle.

The effect of exercise on the binding of glycolytic enzymes to subcellular structures was examined in rainbow trout (Salmo gardneri). Both "burst" and "endurance" type exercise produced an increase of approximately 50% in the percentage of phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase, and 3-phosphoglycerate kinase associated with particulate matter in white skeletal muscle. In contrast, cardiac muscle showed no change in enzyme binding when trout were exercised, suggesting that the effects seen in white muscle are related to an increased anaerobic glycolytic flux in this tissue. Modulation of binding by altered pH or calcium ion concentration was tested: a decrease in pH increased PFK binding to subcellular particles, whereas 2 mM CaCl2 decreased GAPDH binding. These results are discussed with respect to the formation of a glycolytic complex during exercise in trout white muscle.

Binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules.

The interaction of glyceraldehyde 3-phosphate dehydrogenase with microtubules has been studied by measurement of the amount of enzyme which co-assembles with in vitro reconstituted microtubules. The binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules is a saturable process; the maximum binding capacity is about 0.1 mole of enzyme bound per mole of assembled tubulin. Half saturation of microtubule binding sites is obtained at a concentration of glyceraldehyde 3-phosphate dehydrogenase of about 0.5 microM. Glyceraldehyde 3-phosphate dehydrogenase (between 0.1 and 2 microM) induces a concentration-dependent increase a) in the turbidity of the microtubule suspension without alteration of the net amount of polymer formed and b) in the amount of microtubule protein polymers after cold microtubule disassembly. There is a linear relationship between the intensity of the glyceraldehyde 3-phosphate dehydrogenase-induced effects and the amount of microtubule-bound enzyme. The specificity of the association of glyceraldehyde 3-phosphate dehydrogenase to microtubules has been documented by copolymerization experiments. Assembly-disassembly cycles of purified microtubules in the presence of a crude liver soluble fraction results in the selective extraction of a protein with an apparent molecular weight of 35,000 identified as the monomer of glyceraldehyde 3-phosphate dehydrogenase by peptide mapping and immunoblotting. In conclusion, microtubules possess a limited number of binding sites for glyceraldehyde 3-phosphate dehydrogenase. The binding of the glycolytic enzyme to microtubules shows a considerable specificity and is associated with alterations of assembly and disassembly characteristics of microtubules.

A comparative study of the binding of aldolase and glyceraldehyde-3-phosphate dehydrogenase to the human erythrocyte membrane

A high percentage (generally ~60%) of the aldolase-binding capacity of isolated human erythrocyte membranes was lost either during storage of the membranes at 4 °C in 10 m m imidazole-1 m m MgCl 2-10 m m 2-mercaptoethanol, pH 6.8, for 24–48 h, or during brief heating at 25–45 °C. In contrast, relatively little (generally ~25%) of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) binding capacity of the membranes was lost under these same conditions. Binding of both aldolase and G3PDH occurred in (at least) two kinetically distinguishable phases, with a rapidly equilibrating and a slowly equilibrating process being noted. The major portion of the aldolase binding was of a heat-labile rapidly equilibrating type, while the major portion of the G3PDH binding was of a heat-stable slowly equilibrating type. Binding of both aldolase and G3PDH was inhibited by P i in the presence of Mg 2+, with dianionic P i being the effective species. Elution of peripheral proteins, including erythrocyte actin (Band 5), did not cause a decrease in binding of either enzyme that could be attributed to the loss of peripheral proteins. Binding of G3PDH had a markedly detrimental effect on binding of aldolase, but the reverse was not true. Based on this lack of mutual competition as well as the differences in stability of the aldolase and G3PDH binding sites, it is concluded that these enzymes do not bind to a single molecular feature on the erythrocyte membrane.