Rabbit Muscle Glyceraldehyde 3-phosphate Dehydrogenase Research Articles

Mechanism of nicotinamide-adenine dinucleotide binding to rabbit muscle glyceraldehyde 3-phosphate dehydrogenase.

Mechanism of nicotinamide-adenine dinucleotide binding to rabbit muscle glyceraldehyde 3-phosphate dehydrogenase.

Mechanism of alkylation of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMechanism of alkylation of rabbit muscle glyceraldehyde 3-phosphate dehydrogenaseSidney A. Bernhard and Ron A. MacQuarrieCite this: Biochemistry 1971, 10, 13, 2456–2466Publication Date (Print):June 1, 1971Publication History Published online1 May 2002Published inissue 1 June 1971https://doi.org/10.1021/bi00789a005RIGHTS & PERMISSIONSArticle Views97Altmetric-Citations56LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

The Nucleotide and Acyl Group Content of Native Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase

Abstract Glyceraldehyde 3-phosphate dehydrogenase (GP-dehydrogenase) isolated from rabbit muscle by the procedure of Ferdinand (Biochem. J., 92, 578 (1964)), has two previously unreported properties. 1. The coenzyme-binding sites usually filled with NAD+ in native GP-dehydrogenase contain instead the NAD+ degradation product, ADP-ribose. 2. Several of the four active sites contain covalently bound 3-phosphoglyceroyl group. The enzyme isolated by the Ferdinand procedure is in reality the acyl enzyme intermediate in the physiological oxidative phosphorylation of glyceraldehyde 3-phosphate. The occurrence of ADP-ribose appears to be a consequence of the isolation procedure in vitro, but the recovery of acyl enzyme suggests that GP-dehydrogenase is in an acylated state in vivo. The data required to estimate the sarcoplasmic concentrations of NAD+, NADH, the glycolytic intermediates, and the glycolytic enzymes are available in the literature. These estimates and the known effect of acylation on the GP-dehydrogenase-NAD+ affinity suggest two physiological regulatory roles for acyl-GP-dehydrogenase in muscle. One function is the regulation of coenzyme activities and oxidation-reduction potential as the affinity of enzyme for NAD+ changes with degree of acylation, itself dependent on the metabolic state of the tissue. The other is the buffering by acyl enzyme of the more dilute glycolytic intermediates during rapid changes in metabolic state. Although these roles must be considered hypothetical, the finding in skeletal muscle of levels of acylated GP-dehydrogenase much higher than those of many glycolytic intermediates should lead to revision of the accepted glycolytic pathway to include 3-phosphoglyceroyl-GP-dehydrogenase as a significant metabolite.

Nicotinamide Adenine Dinucleotide As an Active Site Director in Glyceraldehyde 3-Phosphate Dehydrogenase Modification

Abstract Multivalent anions, sulfate, phosphate, or arsenate, selectively decrease the rate of alkylation of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by both iodoacetamide and iodoacetate. N-Acetylation of the lysine residue that in the apoenzyme neighbors the essential cysteine residue does not eliminate this anion effect, suggesting that an alternative unique enzyme site is responsible for binding these anions. NAD+ (or 3-acetylpyridine adenine dinucleotide) at high concentration strikingly reverses this effect with iodoacetate; with iodoacetamide a further retardation in alkylation rate is observed. It is proposed that binding of NAD+ limits access of iodoacetamide or other uncharged reagents to the active site sulfhydryl group and simultaneously produces, via the pyridinium cation, a site capable of binding iodoacetate or any other negatively charged reagent. The role of NAD+ in acyl phosphatase and p-nitrophenyl hydrolase activities is considered in this context. The dependence on coenzyme concentration of the alkylation reaction for both reagents was determined with apoenzyme in various media. The results at low NAD+ concentrations in the presence of a multivalent anion indicate that a given coenzyme molecule facilitates, by means of the specific coulombic interaction, carboxymethylation (and not carbamylmethylation) at each of the sites to which it is bound during the reaction period. Finally the possible use of NAD+ (NADP+) as an active site director is commented upon in terms of modifying enzymatic behavior in vivo.

The Properties of Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase Labeled with a "Reporter Group"

Abstract A group, 2-acetamide-4-nitrophenol, has been incorporated at the active site sulfhydryl group of each of the four subunits of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. Changes in the spectrum of this group are produced by changes in pH, addition of DPN and DPNH, or addition of adenosine derivatives including adenosine diphosphoribose, ATP, AMP, and cyclic AMP. Below pH 7.8 the environment of the reporter group is apparently more polar than water while at higher pH there is a shift of the group to a less polar environment. This pH-dependent transition is consistent with the removal of a proton from a neighboring ionized residue with a pK of about 8.5 and is qualitatively similar to the effects observed with reporter-labeled chymotrypsin. Binding of DPN or DPNH also produces a shift of the reporter group to a nonpolar environment, although the changes are different for the two compounds. The alterations in Kdis, subunit interactions, and spectral properties of the reporter-labeled enzyme suggest that the perturbation of the reporter group is due to changes in conformation of the enzyme. The other adenosine compounds are also shown to bind to the enzyme and produce a shift in the spectrum of the reporter group. ATP is unique in that it is tightly bound to two sites on the enzyme but does not compete effectively with DPN. The properties of the reporter-labeled enzyme appear to correspond to those of the normal acyl enzyme intermediate and thus it provides a useful tool for studying the properties of this intermediate.

Reversible Dissociation of Tetrameric 7.4 S Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase into 4.4 S Dimers by Ammonium Sulfate and into 3.2 S Monomers by KCl

Abstract At 0°, native tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (7.4 S) undergoes a reversible inactivation and dissociation into 4.4 S dimers or 3.2 S monomers, depending upon the species of salt present and upon the protein concentration. These dissociation processes have been studied using sucrose density gradient centrifugation. With 0.10 mg per ml of enzyme in 0.15 m ammonium sulfate, 4.4 S dimers predominate; with 0.05 mg per ml of enzyme in 0.15 m KCl, 3.2 S monomers predominate. At other protein concentrations in the two systems, mixtures are produced. KCl gives greater dissociation at all protein concentrations. It also destabilizes at high salt concentrations (1.0 to 2.5 m). In contrast, destabilization in ammonium sulfate is maximal at 0.15 m; 0.5 and 1.0 m ammonium sulfate stabilize against dissociation essentially completely. The native enzyme in the absence of salts shows very slow, very slight dissociation into dimers. Inactivation and dissociation do not occur at 23° in 0.15 m ammonium sulfate and only slightly in 0.15 m KCl, and then only at very low protein concentrations. Inactivation and dissociation are reversed by warming to 23° and greater than 80% of the activity is recovered. The reversible dissociation and inactivation occur at both pH 7.0 (0.1 m imidazole) and at pH 9.5 (0.2 m glycine). A reducing agent (0.1 m β-mercaptoethanol) has to be present for activity to be recovered. These data prove that this enzyme at 0° exists as an equilibrium mixture which favors association to tetramers at high protein concentrations and dissociation into dimers or monomers or both at low protein concentrations. The dimers and monomers appear to be fairly compact.

Function and role of NAD + in mechanism of action of rabbit-muscle glyceraldehydephosphate dehydrogenase

1. 1. NAD + is required for the oxidation of NADH by 1,3-diphosphoglycerate or acetyl phosphate. 2. 2. The maximal rate of acetyl phosphate reduction by NADH or APADH is reached when about 3 moles NAD + per mole enzyme are added. Larger amounts of NAD + inhibit with a K i (NADH as substrate) equal to 45 μM. 3. 3. The NAD-binding site with the lowest affinity for NAD + appears to be the most active in the dehydrogenase reaction. Under optimal conditions the E-(NAD) 3 complex is the catalytically active enzyme. 4. 4. The E-(NAD) 3 complex appears also to be the form with maximal catalytic activity in the transferase reactions. 5. 5. NAD + can be replaced by APAD + in both the dehydrogenase and arsenolysis reactions.

Reversible Dissociation of Tetrameric Rabbit Muscle Glyceraldehyde 3-Phosphate Dehydrogenase into Dimers or Monomers by Adenosine Triphosphate

Abstract Tetrameric 7.5 S rabbit muscle glyceraldehyde 3-phosphate dehydrogenase is dissociated by ATP at 0° into 4.5 S dimers or 2.8 S monomers, depending upon the protein concentration. Dissociation into two peaks is observed at protein concentrations as high as 10 mg per ml, and a 4.5 S dimer is produced in increasing quantity from the 7.5 S tetramer as the protein concentration is lowered from 10 to 2 mg per ml. At 1 mg per ml, a single 4.2 S dimer peak is observed, and at 0.1 mg per ml, a single 2.8 S monomer peak is observed. Between 1.0 and 0.1 mg per ml, only single peaks with sedimentation coefficients intermediate to those of the dimer and monomer are observed, suggesting a fairly rapidly equilibrating mixture of 2.8 S monomers and 4.5 S dimers in varying amounts. The observed inactivation is only slight at temperatures down to 12°, but the equilibrium is shifted fairly strongly toward inactive subpolymers at 7° and apparently is shifted completely to inactive monomers at 0° at 0.05 mg per ml of enzyme. The dissociation has practically no pH dependence in the range of 6.5 to 9.5, but it is somewhat greater and faster at pH 5.5 than at all others studied. Inactivation and dissociation are reversed by rewarming the enzyme to 23° and removing the ATP. Ammonium sulfate, 1 m, or 2 mm NAD protects against inactivation and dissociation. Dissociation is faster with ATP than with other adenine nucleotides and much faster and more pronounced with ATP than with GTP, UTP, CTP, FAD, and CoA.

Binding of NAD + to rabbit-muscle glyceraldehydephosphate dehydrogenase as studied by optical rotatory dispersion and circular dichroism

1. 1. The circular-dichroism spectra of both charcoal-treated and NAD +-containing glyceraldehydephosphate dehydrogenase from rabbit muscle were determined from 200 mμ to 500 mμ. 2. 2. The difference in the optical rotatory dispersion of the holoenzyme and the apoenzyme was calculated from the circular-dichroism spectra by the Kronig-Kramers transformation. 3. 3. The change in the Moffitt-Yang parameter, b 0, and the change in molecular ellipticity at 350 mμ of the enzyme were followed as a function of added NAD +. The curves obtained gave a linear increase up to 2 molecules of NAD + per molecule of enzyme, and an intersection point at 3.0 molecules NAD + per molecule enzyme. 4. 4. By following the change in molecular ellipticity at 330 mμ on addition of 3-acetylpyridine-adenine dinucleotide, a similar result was obtained as with NAD +. 5. 5. Circular dichroism curves with a maximum at 330 mμ were found both with the enzyme-NADH complex and the enzyme-NAD + complex treated with iodoacetate. 6. 6. Since binding of the enzyme with NAD + has no effect on the optical rotatory dispersion or the circular dichroism below 250 mμ, it is concluded that the apo- and holoenzyme do not differ in overall protein conformation. The changes found above 250 mμ are at least partly due to extrinsic bands of the NAD +. 7. 7. The broad band between 300 and 450 mμ in the circular-dichroism spectrum of holoenzyme is probably composed of at least two components: (i) the charge-transfer complex between NAD + and the reactive -SH group of the protein; (ii) an interaction between the adenine moiety and the enzyme.

The reaction between NAD + and rabbit-muscle glyceraldehydephosphate dehydrogenase

1. 1. The A 280 mμ : A 260 mμ ratio of NAD +-free rabbit-muscle glyceraldehydephosphate dehydrogenase was found to be 2.00–2.03. 2. 2. Ultracentrifugation showed that 4 moles of NAD + may be bound to the enzyme per mole (mol. wt. 145 000). The first two molecules are bound stoicheiometrically within the experimental error ( K D < 0.05 μM), while the third and fourth molecules are bound with dissociaton constants of 4 and 35 μM, respectively. 3. 3. The first three molecules of NAD + bound to the enzyme cause the formation of a band at 360 mμ of about equal intensity for each molecule. The fourth molecule causes little further increase of absorption at 360 mμ. 4. 4. A plot of the rate of reduction of NAD + by glyceraldehyde in the presence of arsenate against the NAD + concentration shows a sharp break in the curve at 4 moles NAd + per mole enzyme. 5. 5. Stopped-flow experiments showed that when up to 1 mole of NAD + is added to the enzyme, maximum absorbance increase at 360 mμ is reached within 3 msec. This corresponds to a second-order reaction constant of more than 10 8 M −1·sec −1. With 2 moles of NAD + per mole of enzyme, 81% of the reaction is over in 3 msec, and with 3 or more moles NAD + about 75%. The reaction requires about 1 sec for completion. 6. 6. Prior treatment with NAD + speeds the change of absorbance obtained with subsequent additions of NAD +. The final value obtained, however, is unaltered. 7. 7. For the muscle enzyme under our experimental conditions, a model in which the binding of 1 NAD + molecule to one protomer affects the conformation of a second protomer, either before or after binding with NAD +, appear more appropriate than the allosteric model, which requires that the binding of any one ligand molecule is intrinsically independent of the binding of any other.

Kinetics of the Glyceraldehyde 3-Phosphate Dehydrogenase-catalyzed Hydrolysis of p-Nitrophenyl Acetate

Abstract The kinetics of hydrolysis of p-nitrophenyl acetate by rabbit muscle glyceraldehyde 3-phosphate dehydrogenase has been investigated in aqueous solution at 25° in the pH range 6 to 9. Steady state kinetics was examined under conditions in which substrate is present in great excess over enzyme; pre-steady state kinetics was examined under similar conditions and also when substrate and enzyme are present in approximately equal concentrations. As previously observed, the p-nitrophenolate-time curves were biphasic, exhibiting a rapid exponential reaction, reflecting principally the rate of enzyme acylation, followed by a zero order reaction, reflecting principally the rate of enzyme deacylation. The pH-rate profiles for both acylation and deacylation reactions were carefully examined and found to be similar. In each case, the rate constants increase linearly with increasing hydroxide ion concentration below pH 7.5 and then level off with increasing pH in a manner consistent with the ionization of single groups. Thus, kinetically, acylation depends on a basic group, the conjugate acid of which has a pKa near 8.1, and deacylation depends on a similar group, with pKa near 8.7. The number of moles of p-nitrophenolate released per mole of enzyme in the exponential phase of the reaction is a function of pH, rising from about 2.5 to 3.0 below pH 8 to 5.5 above pH 8. Under steady state conditions, an extraneous reaction was noted at very high substrate concentrations; that is, the rate law under these conditions has the form v = k3E0 + kiiiE0S0. For enzyme acylation with p-nitrophenyl acetate, the magnitude and pH dependence of the rate constants are consistent with unaided attack of a crucial sulfhydryl anion on the ester. In contrast, for deacylation, the magnitude and pH dependence of rate constants require the involvement of a second catalytic entity of the enzyme.

A Study of the Reaction of Glyceraldehyde with Glyceraldehyde 3-Phosphate Dehydrogenase

Abstract Kinetic data indicate that rabbit muscle glyceraldehyde 3-phosphate dehydrogenase possesses two types of diphosphopyridine nucleotide-binding sites. The actual chemical reaction (oxidation-reduction) occurs at tight sites. Binding of DPN to loose sites activates the reaction at the tight sites. These results are consistent with the concept that the enzyme is double headed, with oxidation-reduction heads (which contain the tight sites) and transfer heads (which contain the loose sites). Light scattering data are consistent with the concept that (in the absence of arsenate or phosphate) the enzyme can exist as a dimer (with an apparent weight average molecular weight of 2.8 x 105) which can dissociate upon dilution into monomers (with molecular weights of 1.4 x 105). Dimerization is inhibited by high concentrations of arsenate (0.05 m). This inhibition is overcome by adding DPN to the loose sites. The monomer is unreactive in the absence of arsenate and is markedly activated by adding DPN to the loose sites. The dimer is reactive in the absence of arsenate and is only slightly activated by adding DPN to the loose sites. A kinetic mechanism is proposed which agrees with these and most other observations. This mechanism is sequential with the addition of DPN and then the aldehyde to the oxidation-reduction head. This is followed by the addition of arsenate and then DPN to the transfer head. The mech anism of the reaction with glyceraldehyde and glyceraldehyde 3-phosphate seems to be the same.

The localization of glyceraldehyde 3-phosphate dehydrogenase in kidney tissue by means of fluorescent antibody

1. 1. Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase, highly purified by recrystallization, has been used as an antigen in guinea pigs. Antisera have been obtained which readily precipitate the enzyme and inhibit its activity. 2. 2. An immunochemical evaluation of the precipitability of this preparation with antisera revealed that after simple diffusion in agar gel, pH 7.2, and during electrophoresis in barbital buffer, pH 8.6, the enzyme dissociates into two components each having a different motility. Separation and immunochemical evaluation of these components have shown that both are enzymatically active and are inhibited by the same antisera. It appears, therefore, that the appearance of multiple precipitins in gel and in saline dilutions of the antigen is not due to heterologous contaminants. 3. 3. The localization of the enzyme in the rat kidney by means of fluorescent antibody technique has been described.