Rabbit Muscle Glyceraldehyde 3-phosphate Dehydrogenase Research Articles

Comparisons of Phosphofructokinases-1 from Rabbit, Chicken, and Fish

Background: Previous studies suggested that glycogen storage was facilitated by ascorbate inhibition of phosphofructokinase-1 (PFK-1) in resting mammalian muscle; these studies showed that purified PFK-1 from fish or chicken muscle has properties similar to PFK-1 from mammalian muscles. Materials and Methods: The enzymes utilized in the assay systems came from Sigma-Aldrich Co. Rabbit (Oryctolagus cuniculus) muscle G-actin (A 2522) was free of aldolase, LDH (EC 1.1.1.28) or AK (EC 2.7.4.3) activity and rabbit muscle aldolase (EC 4.1.2.13) was free of AK (EC 2.7.4.3) and LDH activity. Rabbit muscle PFK-1 (RPFK-1), chicken (Gallus gallus) muscle PFK- 1 (CPFK-1) and Pacific red snapper (Lutjanus peru) muscle PFK-1 (FPFK-1) used in these tests were prepared from frozen tissues with modifications of a method. AK, LDH, and aldolase activity were absent in purified FPFK- 1 and CPFK-1 preparations. Results: It can be shown that the following enzymes associated with glycolysis are not inhibited by 0.1 M ascorbate under our conditions: rabbit muscle aldolase (EC 4.1.2.13); rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12); rabbit muscle phosphoglucose isomerase (EC 5.3.1.9); rabbit muscle pyruvate kinase (EC 2.7.1.40); yeast hexokinase (EC 2,7.1.1); and yeast 3-phosphoglyceric phosphokinase (EC 2.7.2.3). Rabbit muscle enolase (EC4.2.1.11) is inhibited under our conditions. Conclusion: In summary, FPFK-1 and CPFK-1, possess characteristics and behaviors similar to RPFK-1, e.g., losses of activities due to dilutions and protections of some of these activity losses by rabbit muscle aldolase. These interactions of a mammalian aldolase with fish and a bird PFK-1’s suggests a conservative evolutionary relationship among aldolases and PFK-1’s.

Evidence for thiol/disulfide exchange reactions between tubulin and glyceraldehyde-3-phosphate dehydrogenase.

While thiol redox reactions are a common mechanism to regulate protein structure and function, protein disulfide bond formation is a marker of oxidative stress that has been linked to neurodegeneration. Both tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contain multiple cysteines that have been identified as targets for oxidation to disulfides, S-nitrosation and S-glutathionylation. We show that GAPDH is one of three prominent brain microtubule-associated proteins (MAPs), in addition to MAP-2 and tau, with reactive cysteines. We detected a threefold to fourfold increase in tubulin cysteine oxidation by hydrogen peroxide in the presence of rabbit muscle GAPDH by 5-iodoacetamidofluorescein labeling and by Western blot detection of higher molecular weight inter-chain tubulin disulfides. In thiol/disulfide exchange experiments, tubulin restored ∼50% of oxidized GAPDH cysteines and the equilibrium favored reduced GAPDH. Further, we report that oxidized GAPDH is repaired by the thioredoxin reductase system (TRS). Restoration of GAPDH activity after reduction by both tubulin and the TRS was time-dependent suggesting conformational changes near the active site cysteine149. The addition of brain MAPs to oxidized tubulin reduced tubulin disulfides and labeling of MAP-2 and of GAPDH decreased. Because the extent of tubulin repair of oxidized GAPDH was dependent on buffer strength, we conclude that electrostatics influence thiol/disulfide exchange between the two proteins. The novel interactions presented herein may protect GAPDH from inhibition under oxidative stress conditions.

Ligand Binding Energy and Enzyme Efficiency from Reductions in Protein Dynamics

Tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) binds successively four molecules of its cofactor (NAD+) with affinities of ca 10(11) M(-1), 10(9) M(-1), 10(7) M(-1), and 10(5) M(-1). The reduction in the dynamics of the protein is greatest upon binding the first NAD+ molecule. Smaller reductions then occur upon binding the second and third NAD+ molecules, and the fourth NAD+ molecule binds without dynamic change. Reduction of the GAPDH dynamics, with consequent improvements in its internal bonding, can account for the increase in NAD+ binding affinity from 10(5) M(-1) to 10(11) M(-1). Evidence is provided that comparable fractions of the binding energy of other ligands, and of the catalytic efficiency of enzymes, may be derived in the same way.

Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae

Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49–68 and 69–90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49–68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.

Rabbit muscle GAPDH: non-phosphorylating dehydrogenase activity induced by hydrogen peroxide

Incubation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with micromolar hydrogen peroxide concentrations does not alter the catalytic properties of GAPDH in the reaction of oxidative phosphorylation of glyceraldehyde-3-phosphate, but endows the enzyme with the ability to catalyze the reaction in the absence of inorganic phosphate, producing NADH and 3-phosphoglycerate. The reaction is supposed to occur as a result of intramolecular acyl transfer from Cys-149 to a sulfenic acid form of Cys-153, followed by hydrolysis of the intermediate. The ‘mildly oxidized’ form of the enzyme can be easily converted back to the form unable to catalyze glyceraldehyde-3-phosphate oxidation in the absence of phosphate, by the addition of thiols.

A solid support for affinity chromatography that covalently binds thiol groups via a cleavable connector arm

Preparation of an agarose derivative (MPE-agarose) containing a maleimido group which is attached to agarose via a cleavable phenyl ester linkage is described. MPE-agarose was shown to react with the thiol groups in glutathione, bovine serum albumin, bovine hemoglobin, and yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. Treatment of the resulting agarose-linked compounds for 10 min with 1 m hydroxylamine (pH 7) resulted in the cleavage of the phenyl ester linkage, and release of the maleimido derivative of the compound from the gel. In the case of hemoglobin and glyceraldehyde 3-phosphate dehydrogenase noncovalent interactions between the gel and the released protein lowered the amount of protein which dissolved in the hydroxylamine solution upon cleavage of the phenyl ester linkages. Noncovalently absorbed protein could be removed from the gel, however, by washing the gel with 2 m guanidine hydrochloride after treatment with hydroxylamine. Derivatives of MPE-agarose should prove useful in affinity chromatography and immunoabsorption where it is difficult to elute material bound to conventional affinity supports.

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD + and NADH binding

1. 1.|The binding parameters for NADH and NAD + to rabbit-muscle glyceraldehyde-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the fluorescence of the protein and the NADH. 2. 2.|The fact that the degree of protein fluorescence quenching by bound NAD + or NADH, excited at 285 nm and measured at 340 nm (‘blue’ tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentrations of sites, if linearity is assumed. 3. 3.|This is also the case for NADH, but not for NAD +, when the protein fluorescence is excited at 305 nm and measured at 390 nm (‘red’ tryptophans). 4. 4.|The binding of NAD + can be described by a model in which the binding of NAD +, via negative interaction within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 μM at low saturation and 0.18 μM for the holoenzyme. 5. 5.|The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 μM and of the holoenzyme 1.0 μM. 6. 6.|The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD + or NADH. 7. 7.|The binding of NAD + to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD + to this form of the enzyme is in agreement with the K i calculated from product inhibition by NAD + of the reductive dephosphorylation of 1,3-diphosphoglycerate.

The interaction of adenine with its binding site in rabbit muscle glyceraldehyde 3-phosphate dehydrogenase studied by fluorescence decay

The fluorescence decay mechanism of 1, N 6-ethenoadenosine diphosphoribose bound to rabbit muscle glyceraldehyde 3-phosphate dehydrogenase markedly differs from that of the intact coenzyme analog (εNAD +) bound to the same enzyme. In the latter case the fluorescence is partially quenched by interactions between the ethenoadenine ring and amino acid residues in its binding site. Binding of the nicotinamide moiety of the coenzyme thus affects the relative orientation of the adenine ring within its binding site leading to the quenching interactions. The interactions of the adenine group with its binding site induce conformational changes in the enzyme which affect the binding of additional coenzyme molecules. The nicotinamide base thus determines, indirectly, the negative cooperativity found in NAD + binding.

Preparation and properties of rabbit-muscle glyceraldehyde-phosphate dehydrogenase with equal binding parameters for the third and fourth NAD + molecules

1. 1. A method of preparing rabbit-muscle glyceraldehyde-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) is described which yields a preparation differing in important respects from those previously described and resembling the enzyme isolated from sturgeon muscle. 2. 2. Direct binding measurements at 25°C by equilibrium gel filtration fit dissociation constants for the first two molecules that are too low to be measured by this technique and 0.9 μM for the third and fourth molecules. The dissociation constant of the fourth molecule is much lower than that previously reported for the rabbit-muscle enzyme. 3. 3. In contrast to previous results with the rabbit-muscle enzyme, the increase in absorbance at 360 nm between three and four molecules of NAD + bound to the enzyme was, within experimental error, the same as that with each of the first three molecules. 4. 4. Data on the quenching of the protein fluorescence by NAD + at 15°C at different enzyme concentrations closely fit dissociation constants of 0.028 μM for the first two molecules and 0.27 μM for the third and fourth molecules.

Synthesis of a reactive ATP analog and its preliminary application as an affinity labeling reagent.

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP.PCP, was synthesized in an attempt to covalently label the binding sites for adenine nucleotides, especially ATP, of various enzymes which utilize adenine nucleotides as substrates, cofactors, inhibitors or allosteric effectors. This reagent rapidly inactivated rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD), myokinase (MK), and creatine kinase (CK) under very mild conditions. Adenine nucleotide substrates prevented the inactivation. In the case of GPD, complete inactivation was observed when 1 mol of the reagent per mol of enzyme subunit was incorporated into the enzyme. These results indicate that the present ATP analog may be useful as an affinity labeling reagent for various adenine nucleotide-dependent enzymes.

Affinity labeling of adenine nucleotide-related enzymes with reactive adenine nucleotide analogs. I. Affinity labeling of glyceraldehyde 3-phosphate dehydrogenase and myokinase with a reactive AMP analog.

Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD) and myokinase (MK) were rapidly inactivated by a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP, under mild conditions. Complete inactivation was observed when 4 and 0.3 mol of the reagent with respect to enzyme were reacted with GPD and MK, respectively. The inactivation of both enzymes were favored at higher pH and the enzymes were protected by addition of adenine nucleotide substrate. Modified GPD or MK had no affinity for AMP-Sepharose, in contrast to the native enzymes. From these results, the inactivation of GPD and MK by the reactive AMP analog can be regarded as an affinity labeling. The posibility that the present AMP analog may be used as a general affinity labeling reagent for various adenine nucleotide-related enzymes is discussed based on the results obtained.

Ion-Affinity Electrophoresis. Preparation and Properties of Phosphorylated Polyacrylamede Gels

Strong, pliable polyacrylamide gels containing covalently bound phosphate groups have been formed by radical-initiated copolymerization of acrylamide and a phosphorylated, N-substituted derivative of acrylamide. Under conditions of disc electrophoresis, the gels provide enhanced resolution in the separation of hemoglobin A from S, and separate two forms of yeast glucose 6-phosphate dehydrogenase and rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. Preliminary evidence suggests that the improved resolution is due to ionic adsorption of the proteins to immobilized phosphate groups, implying that the gels may function as supports for electrophoretically powered separations involving ion-exchange or affinity chromatography.

Analysis of the allosteric basis for positive and negative co-operativity and half-of-the-sites reactivity in yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenase

A general model of co-operativity, which combines the concepts of preferential ligand binding, molecular constraints, and nearest-neighbor constraints, is used to analyze quantitatively the co-operative properties of yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. The model is found to fit equilibrium dialysis and spectrophotometric measurements of NAD binding, measurements of the NAD dependence of proteolytic inactivation, and measurements of the NAD dependence of enzyme activity with consistent sets of parameter values. It is concluded that this enzyme has two quaternary conformations and that the change from the quaternary conformation of the apoenzyme to the quaternary conformation of the holoenzyme occurs roughly when the first NAD molecule is bound; this change is responsible for the fact that in the yeast enzyme the second NAD molecule has a higher binding affinity than the first and that the resistance of the yeast and rabbit muscle enzymes to proteolysis increases sharply when the first one or two NAD molecules bind. In addition, within the quaternary conformation of the holoenzyme, NAD binding at different sites is coupled by a tetrahedral (rather than square or dimer) pattern of interactions; these interactions are responsible for negative co-operativity in glyceraldehyde 3-phosphate dehydrogenase. Pairwise asymmetry (induced or pre-existing) is excluded as the basis for the co-operativity observed in NAD binding. Analysis of the temperature-dependence of model parameters shows that the change from the apo to the holo quaternary conformation is a strongly endothermic process, accompanied by an increase in entropy, whereas binding of NAD to the enzyme in the holo quaternary conformation is a strongly exothermic process, accompanied by a decrease in entropy. The enthalpy and entropy associated with nearest-neighbor constraints change with temperature, suggesting that an effector is present which influences the strength of these constraints. When the enzyme is in the quaternary conformation characteristic of the apoenzyme, it is essentially inactive. Half-of-the-sites reactivity can be explained by supposing that modification of the cysteine-149 sulfhydryl groups stabilizes an inactive quaternary conformation of the enzyme. In contrast to other proposed explanations of half-of-the-sites reactivity, the explanation given here does not assume molecular asymmetry or direct interactions between sites, and is therefore also applicable to enzymes which show no negative co-operativity. Numerous phenomena related to half-of-the-sites reactivity are also shown to be consistent with the present model. Whereas in the past different models have often been used to explain different properties of glyceraldehyde 3-phosphate dehydrogenase the present model consistently explains a very wide variety of data.

Purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by gel filtration chromatography

A procedure is presented for the large-scale purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12). Sephadex G-100 chromatography was more effective than repeated recrystallizations for removing heme impurities, hence these proteins appear to cocrystallize. The column purified enzyme has full enzymatic activity according to dehydrogenase, esterase, and acetyl phosphatase assays.

Half-of-the-sites and all-of-the-sites reactivity in rabbit muscle glyceraldehyde 3-phosphate dehydrogenase

The behavior of rabbit muscle apoglyceraldehyde 3-phosphate dehydrogenase towards alkylating agents depends on the chemical nature of the modifying reagent. Two classes of reagents were found, those yielding half-of-the-sites reactivity and those resulting in all-of-the-sites reactivity. On the basis of results obtained in this study and our previous investigations on the nature of enzyme-coenzyme interactions, a molecular explanation for this phenomenon is offered. It is suggested that interactions of the alkyl group of the modifying reagent at the nicotinamide subsite of the NAD + site or the glyceraldehyde 3-phosphate site are not transmitted to other subunits and all-of-the-sites reactivity results. The reagents that contain alkyl groups which interact with the adenine subsite region of the NAD + site induce a conformational change that is transmitted to a vacant subunit across one of the three sets of isologous intersubunit binding domains within the tetramer, thus inducing half-of-the-sites reactivity. The pattern of half-of-the-sites reactivity indicates that the enzyme tetramer functions as a dimer of dimers. It is suggested that the interaction that leads to half-of-the-sites reactivity occurs between the hydrophobic alkyl group and the hydrophobic trimethylene aide chain of Lys 183 originating from the neighboring subunit across the intersubunit domain, or with other amino acid residues.

The Interaction of Glyceraldehyde 3-Phosphate Dehydrogenase with Human Erythrocyte Membranes

Abstract Human erythrocyte ghosts retain most of the enzyme glyceraldehyde 3-phosphate dehydrogenase bound to the membrane. The bound dehydrogenase, 3 x 105 molecules per ghost, can be eluted with increasing concentrations of salt, with 50 % elution occurring at 0.16 m NaCl, pH 7.5. Exogenous glyceraldehyde phosphate dehydrogenase can bind to the membrane. Two types of binding sites have been detected. The high affinity sites bind a maximum of 3 x 105 molecules of dehydrogenase per ghost with a Kd less than 10-8 m for the enzyme obtained from either erythrocytes or rabbit muscle. The low affinity sites bind up to 63 x 105 molecules per ghost with Kd greater than 10-7 m. Enzyme bound to the membrane in this way can be re-eluted with NaCl, indicating freely reversible interaction between the enzyme and membrane. Kant and Steck (J. Biol. Chem. (1973) 248, 8457–8464) have reported reversible binding of erythrocyte glyceraldehyde phosphate dehydrogenase with ghost membranes; however, their conditions did not permit the demonstration of two types of binding sites. There is specificity in the enzyme-membrane interactions. Both the erythrocyte and rabbit muscle glyceraldehyde phosphate dehydrogenase bind to the high and low affinity sites, but yeast enzyme interacts only weakly with the membrane. A large amount of yeast enzyme (62 x 105 molecules per ghost) binds with a Kd of 10-4 m, binding which resembles the interaction of cytochrome c with this membrane. Ghosts prepared and sealed in the presence of MgSO4 bind very little glyceraldehyde phosphate dehydrogenase and no binding occurs with high affinity. This result agrees with the conclusion of Kant and Steck that the dehydrogenase specific sites are located on the cytoplasmic surface of the membrane. Ghosts incubated at 37° in NaCl become partially sealed. Such ghosts also bind the dehydrogenase both at the high and low affinity sites. However, at each type of site part of the bound enzyme is cryptic, i.e. its activity is masked unless a detergent, Triton X-100, is present. It appears that under these conditions there is an allotopic effect on some of the dehydrogenase when it interacts with the membrane. The number of high affinity glyceraldehyde phosphate dehydrogenase binding sites on the ghost membrane is similar to that of other known membrane proteins of erythrocytes and about equals the average number of the dehydrogenase molecules in the intact erythrocyte. These facts, together with the high specificity and high affinity, suggest that the interaction between this enzyme and the membrane may have a physiological function. Because both the enzyme and membrane are well characterized they form a valuable model system for examining enzyme-membrane interactions in vitro.

Binding of NAD + to rabbit-muscle glyceraldehydephosphate dehydrogenase. The use of [formula omitted] chloride as a spectral and conformational probe

Addition of N- methylnicotinamidium chloride to rabbit-muscle glyceraldehydephosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) results in the appearance of a charge-transfer absorption spectrum, with an apparent maximum around 327 nm, that is insensitive to the sulfhydryl reagents iodoacetate and p- chloromercurisulfonate . Resolution of this band and of the Racker band, resulting from the binding of NAD + to the enzyme, gives two overlapping Gaussian absorption bands with maxima at the same wavelength (324 and 369 nm) but with different relative intensities for the two complexes. From these data it is concluded that tryptophan is involved as the electron-donating group in the charge-transfer absorption band of the enzyme-NAD + complex. The different relative intensities of the two overlapping Gaussian absorption bands are attributed to dissimilarities in the relative orientation of the nicotinamidium ion of NAD + and N- methylnicotinamidium chloride, respectively, with respect to the tryptophan molecule involved. By using the charge-transfer absorption band of the enzyme-N- methylnicotinamidium complex as a conformational probe, it is shown that the effect on the protein conformation, caused by the binding of the first two NAD + molecules to the enzyme, is different from that brought about by the binding of the third and fourth NAD + molecule.

The effect of substrates, effectors, acetyl phosphate and iodoacetate on the binding of NAD + and NADH to rabbit-muscle glyceraldehydephosphate dehydrogenase

1. 1. None of the substrates (glyceraldehyde, acetaldehyde, phosphate or arsenate) or effectors (ADP and ATP) tested have an appreciable effect on the binding of NAD + to rabbit-muscle glyceraldehydephosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12). Glyceraldehyde, glyceraldehyde 3-phosphate, arsenate, phosphate, ADP and ATP have also no effect on the binding of NADH. 2. 2. The product, acetyl phosphate, which acetylates the active-centre thiol group, and the inhibitor iodoacetate, which carboxymethylates it, both decrease the affinity of the NAD + molecule for the enzyme. Iodoacetate also decreases the affinity of at least the third and fourth NADH molecule to the enzyme (acetyl phosphate could not be tested with NADH). However, even after acetylation or carboxymethylation, the binding of both NAD + and NADH remains negatively co-operative. 3. 3. The binding constants for NAD + and NADH do not differ appreciably under any of the conditions tested. 4. 4. Acetyl phosphate decreases the absorbance coefficient of the 360 nm band for the third NAD + molecule bound to the enzyme. Between pH 7.0 and 7.5 the coefficient for this molecular becomes equal to that of the fourth NAD + molecule bound to the enzyme. Acetyl phosphate has no effect on the absorbance coefficient of the first, second or fourth molecule of bound NAD +. Iodoacetate abolishe the 360-nm band given by all four NAD + molecules. 5. 5. None of the substrates or effectors tested has any effect on the circular-dichroism band at 335 nm, caused by the binding of NADH to the enzyme. However, this band is completely abolished by iodoacetate. 6. 6. None of the substrates, effectors or product tested has any effect on the degree of quenching of the protein fluorescence, caused by the binding of either NAD + or NADH to the enzyme. Nor does iodoacetate have any effect. The dissociation constants derived in this way are in good agreement with those calculated from direct separation techniques.

Effects of ATP and 2,3-diphosphoglycerate on glyceraldehyde 3-phosphate dehydrogenase activity

Summary We have obtained a cacodylate-dependent system in which ATP at physiological concentrations (10 −4 M) dissociates and inactivates rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD) at 25° C. We have shown that this inactivation and dissociation of GPD is blocked by physiological concentrations of 2,3-diphosphoglycerate (2,3-DPG). These same effects have also been noted for the human erythrocyte GPD. ATP and 2,3-DPG may therefore be of importance in regulation of carbohydrate metabolism, particularly in the red blood cell where high concentrations of ATP and 2,3-DPG are found.

Binding of NAD + and NADH to rabbit-muscle glyceraldehydephosphate dehydrogenase

1. 1. The fourth NAD + molecule bound to glyceraldehydephosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) contributes to the absorbance at 360 nm and the molecular ellipticity at 350 nm, but the absorbance coefficient of the NAD +-enzyme complex is 30% and the molecular ellipticity 40% of that found for the first three NAD + molecules bound. 2. 2. NAD +, present in excess of that sufficient to saturate the four specific binding sites, reacts with the enzyme in such a way as to give what is very probably a charge-transfer complex. 3. 3. Ultracentrifugation and ultrafiltration showed that 4 moles of NADH may be bound to the enzyme. The first two molecules are bound stoicheiometrically within the experimental error ( K rmD < 0.5 μ M ), while the third and the fourth molecules are bound with dissociation constants of 2.5 and 50 μM, respectively. 4. 4. The first three molecules of NADH bound to the enzyme cause the formation of a circular-dichroism band at 335 nm of equal intensity for each molecule. The fourth molecule does not contribute to the molecular ellipticity at 335 nm. 5. 5. NADH inhibits the oxidation of glyceraldehyde by NAD +, in the presence of arsenate, competitively with respect to NAD +. The K i 90 μM, is similar to that of the dissociation constant of the fourth molecule of NADH.