Putative Synaptic Vesicle Nucleotide Transporter Identified as Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract: Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N -ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins of AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.     

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.     

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase: Structural basis for enhanced stability

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is bound to the fibrous sheath of the sperm flagellum through the hydrophobic N-terminal domain of the enzyme molecule. Expression of human GAPDS in E.coli cells yields inactive and insoluble protein. Presumably, the N-terminal domain prevents correct folding of the full-length recombinant enzyme. To obtain GAPDS in a soluble and active form, a recombinant enzyme lacking in 68 amino acids of the N-terminal domain (dN-GAPDS) was expressed in E.coli cells. Purified dN-GAPDS was shown to be a protein of 9.3 nm in diameter (by dynamic light scattering), which is close to the size of the muscle tetrameric glyceraldehyde-3-phosphate dehydrogenase (8.6 nm). The catalytic properties of the protein differed a little from those of the muscle glyceraldehyde-3-phoshate dehydrogenase. However, compared to muscle glyceraldehyde-3-phoshate dehydrogenase, dN-GAPDS exhibited enhanced thermostability (the transition midpoints values are 60.8 and 67.4 °C, respectively) and was much more resistant towards action of guanidine hydrochloride (inactivation constants are 2.45 ± 0.018 and 0.118 ± 0.008 min^? 1, respectively). The enhanced stability of dN-GAPDS is likely to be related to some specific features of the GAPDS structure compared to that of the muscle enzyme: 1) reduced number of solvent-exposed salt bridges; 2) 2 additional buried salt bridges; and 3) 6 additional proline residues in GAPDS meeting the “proline rule”. It is assumed that high stability of the sperm-specific GAPDS is of importance for the efficiency of fertilization.     

Non-functioning chaperonin GroEL stimulates protein aggregation

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be decreased in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The size of individual proteins (GroEL, GAPDH, and the recombinant prion) and aggregates formed after their mixing have been determined by the dynamic light scattering analysis. It was shown that at 25°C, the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the size of the aggregates to 70–80 nm. After blocking of one of the chaperonin active sites by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.     

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.     

Chaperonins induce an amyloid-like transformation of ovine prion protein: the fundamental difference in action between eukaryotic TRiC and bacterial GroEL

Molecular chaperones have been shown to be involved in the processes taking place during the pathogenesis of various amyloid neurodegenerative diseases. However, contradictory literature reports suggest that different molecular chaperones can either stimulate or prevent the formation of amyloid structures from distinct amyloidogenic proteins. In the present work, we concentrated on the effects caused by two molecular chaperonins, ovine TRiC and bacterial GroEL, on the aggregation and conformational state of ovine PrP. Both chaperonins were shown to bind native PrP and to produce amyloid-like forms of ovine PrP enriched with beta-structures but, while GroEL acted in an ATP-dependent manner, TRiC was shown to cause the same effect only in the absence of Mg-ATP (i.e. in the inactive form). In the presence of chaperonin GroEL, ovine PrP was shown to form micellar particles, approximately 100-200nm in diameter, which were observed both by dynamic light scattering assay and by electron microscopy. The content of these particles was significantly higher in the presence of Mg-ATP and, only under these conditions, GroEL produced amyloid-like species enriched with beta-structures. TRiC was shown to induce the formation of amyloid fibrils observed by electron microscopy, but only in the absence of Mg-ATP. This study suggests the important role of the cytosolic chaperonin TRiC in the propagation of amyloid structures in vivo during the development of amyloid diseases and the possible role of the bacterial chaperonin GroEL, located in the intestinal microflora, in the induction of these diseases.     

Immobilization of glyceraldehyde-3-phosphate dehydrogenase by non-covalent binding to specific antibodies and Fab-fragments coupled to Sepharose]

Rabbit antibodies to rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase, as well as monovalent Fab fragments of these antibodies were coupled to CNBr-activated Sepharose 4B. Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was then immobilized on a matrix by non-covalent binding to specific antibodies. Immobilized enzyme retains approximately 90% catalytic activity of the soluble dehydrogenase; pH optimum of activity and the Km value observed are changed as compared to the enzyme in solution. Glyceraldehyde-3-phosphate dehydrogenase immobilized on specific antibodies is shown to undergo adenine nucleotide-induced dissociation into dimers. The immobilized dimeric form of the enzyme thus obtained is catalytically active and capable of reassociating with the dimers of apoglyceraldehyde-3-phosphate dehydrogenase added in solution to the suspension of Sepharose.     

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(2)O(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.     

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.     

Effect of coenzyme on conformational stability of glyceraldehyde-3-phosphate dehydrogenase from muscles of ecto- and endothermic animals]

The stabilizing effect of the coenzyme (NAD) on the structure of glyceraldehyde-3-phosphate dehydrogenase from lamprey and porcine muscles with respect to proteolysis and heat denaturation was studied. The process of heat denaturation was followed by the changes in specific activity of the enzymes; that of proteolysis--by the changes in specific activity and circular dichroism. It was shown that in both cases NAD at saturating concentration exerts a far weaker stabilizing effect on the structure of glyceraldehyde-3-phosphate dehydrogenase from lamprey muscle than on that of the porcine muscle enzyme. The coensyme-dependent stabilization of lamprey muscle glyceraldehyde-3-phosphate dehydrogenase does not differ from that of mammalian muscle enzyme. Possible interrelationship between the phenomenon observed and the molecular mechanism of thermal adaptation in the cold-blooded animals is discussed.     

Somatic and sperm-specific isoenzymes of glyceraldehyde-3-phosphate dehydrogenase: Comparative analysis of primary structures and functional features

The elucidation of the interdependence between structural features and functions of somatic and sperm-specific isoenzymes of glyceraldehyde-3-phosphate dehydrogenase (GAPD and GAPDS, respectively) was the goal of comparative analysis of their primary structures. GAPDS was shown to lack the sequence similar to the atypical nuclear export signal motif (NES) of the somatic isoenzyme GAPD. This finding is confirmed by experimental data on the absence of interaction between GAPDS and antibodies 6C5 recognizing the NES motif in the sequence of GAPD. The lack of NES correlates with functional peculiarities of the sperm-specific enzyme that is tightly bound to the fibrous sheath of the sperm flagellum. The sequences of the two isoenzymes were examined for the short motifs that might participate in apoptosis, endocytosis, and DNA repair. Sites of phosphorylation by different protein kinases have been revealed in both isoenzymes, and their characteristic features are discussed. These observations can serve as the basis for subsequent search for new ways of regulating the two isoenzymes.     

Properties of two high-molecular-mass forms of glyceraldehyde-3-phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity.

Two high-Mr forms of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach leaf can be separated by DEAE-cellulose chromatography. One form, the high-Mr glyceraldehyde-3-phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G.R., Ortwerth, B.J. & Koeppe, O.J. (1970) J. Biol. Chem. 245, 4193-4198]. The other, a glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex, is characterised by possession of latent phosphoribulokinase activity, only expressed following incubation with dithiothreitol. This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high-Mr glyceraldehyde-3-phosphate dehydrogenase, but also of a third subunit, R (40.5 kDa) comigrating with that from the active phosphoribulokinase of spinach. Incubation of the complex with dithiothreitol markedly stimulated both its phosphoribulokinase and NADPH-dependent dehydrogenase activities. This dithiothreitol-induced activation was accompanied by depolymerisation to give two predominantly NADPH-linked tetrameric glyceraldehyde-3-phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high-Mr glyceraldehyde-3-phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the heterotetramer (A2B2). Possible structures suggested for the glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex are (A2B2)2A4R2 or (A2B2)(A4)2R2.     

The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase in situ PMR studies

The kinetics of inhibition of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by iodoacetate were studied in the intact cell and in vitro. The kinetics were determined using ¹H-NMR to follow solvent exchange of ¹H and ²H at the C-2 position of lactate. The exchange occurs via a series of enzyme-catalysed reactions, including that catalysed by glyceraldehyde-3-phosphate dehydrogenase. A direct assay with quenching of the inhibition was also used to check the results. Iodoacetate was shown to act as an active site-directed inhibitor of the dehydrogenase. The enzyme inhibition patterns, which are characterised by a binding step and a kinetic step, are similar in situ and in vitro. Membrane binding, however, was found to alter the inhibition pattern for the enzyme in vitro.     

Protein damage, induced by small amounts of photodynamically generated singlet oxygen or hydroxyl radicals

The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and myoglobin by singlet oxygen and by hydroxyl radicals was investigated. The intrinsic fluorescence of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase decreased rapidly during oxidation, indicating a conformational change of the protein molecules. The free energy of isothermal unfolding in urea solutions was increased by singlet oxygen, but decreased by hydroxyl radical attack. The velocity of refolding of the denatured protein after dilution of the denaturant was increased by exposure to either singlet oxygen or hydroxyl radicals, with one exception: the velocity of refolding of myoglobin, oxidized by singlet oxygen, was strongly decreased. Hydroxyl radicals produced covalently crosslinked protein aggregates and some fragmentation, whereas singlet oxygen produced only crosslinked aggregates with glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase. All oxidized proteins were more susceptible to proteolysis by elastase and proteinase K, as compared to the undamaged proteins. Singlet oxygen-induced crosslinked aggregates were degraded very rapidly by elastase. Hydroxyl radical-induced aggregates of glyceraldehyde-3-phosphate dehydrogenase were also degraded very rapidly by this enzyme, but hydroxyl radical-induced aggregates of alcohol dehydrogenase were resistent to enzymatic degradation. The results indicate that limited protein oxidation may have a pronounced influence on several properties of the protein. The effects vary, however, with varying proteins and with the oxidizing species.     

Immobilized hybrids of glyceraldehyde-3-phosphate dehydrogenase.

Yeast glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) immobilized on CNBr-activated Sepharose 4-B has been subjected to dissociation to obtain matrix-bound dimeric species of the enzyme. Hybridization was then performed using soluble glyceraldehyde-3-phosphate dehydrogenase isolated from rat skeletal muscle. Immobilized hybrid tetramers thus obtained were demonstrated to exhibit two distinct pH-optima of activity characteristic of the yeast and muscle enzymes, respectively. The results indicate that under appropriate conditions the activity of each of the dimers composing the immobilized hybrid tetramer can be studied separately.     

The pentose phosphate pathway and NADP-dependent glycerol-3-phosphate dehydrogenase activity in some tissues of albino rat]

The NADP-dependent glycerol-3-phosphate dehydrogenase activity in liver, heart and skeletal muscle of rat was studied. The activity is found when glyceraldehyde-3-phosphate or ribose-5-phosphate in the presence of ATP are taken as substrates. The data obtained confirm that NADP-dependent glycerol-3-phosphate dehydrogenase exists in skeletal muscle and demonstrate that it is found in heart muscle as well.     

Kinetic evidence for interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase.

The possibility of interaction between purified rabbit muscle aldolase and D-glyceraldehyde-3-phosphate dehydrogenase was studied by rapid kinetic methods, by analyzing the kinetics of the consecutive reaction catalyzed by the coupled enzyme system. The Km of the intermediary product, glyceraldehyde 3-phosphate, produced by aldolase was determined in the coupled reaction for glyceraldehyde-3-phosphate dehydrogenase. Its value corresponds to that of the aldehyde (active) form of glyceraldehyde 3-phosphate, although in the given conditions the aldehyde leads to diol interconversion is faster than the enzymic reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. We suggest that above a certain concentration of the enzymes the glyceraldehyde 3-phosphate produced by aldolase gets direct access to glyceraldehyde-3-phosphate dehydrogenase without participating in the aldehyde leads to diol interconversion which otherwise would occur if the substrate were to mix with the bulk medium.     

Inhibition of chaperonin GroEL by a monomer of ovine prion protein and its oligomeric forms

The possibility of inhibition of chaperonin functional activity by amyloid proteins was studied. It was found that the ovine prion protein PrP as well as its oligomeric and fibrillar forms are capable of binding with the chaperonin GroEL. Besides, GroEL was shown to promote amyloid aggregation of the monomeric and oligomeric PrP as well as PrP fibrils. The monomeric PrP was shown to inhibit the GroEL-assisted reactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The oligomers of PrP decelerate the GroEL-assisted reactivation of GAPDH, and PrP fibrils did not affect this process. The chaperonin GroEL is capable of interacting with GAPDH and different PrP forms simultaneously. A possible role of the inhibition of chaperonins by amyloid proteins in the misfolding of the enzymes involved in cell metabolism and in progression of neurodegenerative diseases of amyloid nature is discussed.     

兔肌3-磷酸甘油脱氢酶的提纯及性质研究

目的:研究兔肌3-磷酸甘油脱氢酶的分离纯化方法及其酶学性质,为测定血清甘油三酯所用酶联试剂的开发提供试验基础和理论依据。方法:通过硫酸铵分级沉淀、DEAE-Sepharose、Blue-Sepharose和羟磷灰石纯化兔肌3-磷酸甘油脱氢酶,利用凝胶过滤和梯度PAGE(5%～15%)法测定酶分子量,采用常规酶学动力学分析方法,考察pH、温度、底物浓度以及部分金属离子与有机化合物对酶促反应的影响。结果 纯化后的兔肌3-磷酸甘油脱氢酶经PAGE(12%)分析为单一条带;酶分子量为115～122 kDa;酶最适温度45℃,最适pH 9;酸碱稳定范围pH6～9,低于45℃时热稳定性好;最适条件下,以3-磷酸甘油和NAD⁺为底物,测得酶的K_m分别为7.4×10^-3mol/L和1.47×10^-4mol/L;Ba²⁺、Mn²⁺、Fe²⁺、Al³⁺、Cu²⁺、Ni²⁺、Ag⁺、Hg²⁺、NaN₃、EDTA对酶有不同程度的抑制作用,Mg²⁺、Ca²⁺、Co²⁺、Zn²⁺有一定程度的激活作用,其中Co²⁺和Zn²⁺对酶的激活作用能达到200%以上,有机化合物NaF对酶的活性没有影响。     

Novel isolation method and structural stability of a eukaryotic chaperonin: the TCP-1 ring complex from rabbit reticulocytes.

In the course of removing a contaminant from preparations of aminoacyl-tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin-agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino-terminal sequencing, and ATP-dependent refolding of rhodanese and actin. Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the "characteristic" ring and four-stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate-containing views are observed. The particle structure is strikingly resistant to physical forces (long-term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KCl or LiCl), and pH changes (9-6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KCl or deoxycholate reduces TRiC folding activity.