Sturgeon Muscle Research Articles

Role of Lysine-183 in d-Glyceraldehyde-3-Phosphate Dehydrogenases. Properties of the N-Acetylated Yeast, Sturgeon Muscle and Rabbit Muscle Enzymes

The acetylation of the ɛNH2 of lysine-183 of rabbit muscle, sturgeon muscle and yeast glyceral-dehyde-3-phosphate dehydrogenases at pH 8.5, by SN acetyl transfer follows a first-order process. Essentially, four lysine residues are modified per enzyme tetramer. When enzymic assays are carried out under saturating conditions of the three substrates (NAD+, glyceraldehyde 3-phosphate and arsenate), the N-acetylated rabbit and sturgeon muscle enzymes show values of V which are 90% and 80% respectively of that of the native enzymes, while for the N-acetylated yeast enzyme it is only 20%. The Km values for the three substrates are not significantly affected by the N-acetylation of the two muscle enzymes. However, the N-acetylated yeast enzyme yields an increased Km for NAD+ only. By spectrophotometric titration at the Racker band, it was shown that each of the three N-acetylated enzymes still binds four NAD+ per tetramer. Spectrofluorimetric titration of the binding of NAD+ to the N-acetylated rabbit muscle enzyme revealed that acetylation of lysine-183 affected essentially the first three macroscopic NAD+ dissociation constants. The fourth macroscopic dissociation constant remained of the same order of magnitude as that found for the native enzyme. However, the calculated microscopic dissociation constants show that the affinities of the second, third and fourth NAD+ are of the same order. This result indicates that acetylation of lysine-183 desensitized the anticooperative binding process of the coenzyme. Furthermore, it was observed that this chemical modification also affected the profile of arsenate activation for the three enzymes. Spectroscopic, ultracentrifugation and thermostability studies failed to show any significant conformational change due to the N-acetylation. In conclusion, our results show that the four lysine-183 residues per tetramer of muscle or yeast glyceraldehyde-3-phosphate dehydrogenase are equally accessible to N-acetylation. Reaction with the yeast enzyme is much slower than that with the muscle enzymes. It is shown further that lysine-183 is implicated neither in the catalytic site nor in the substrate binding site. However, this residue seems to play an important role in the area of contact between the two adjacent subunits through which flows conformational information incidental to the binding of NAD+ or arsenate to the protein. This result is in agreement with X-ray crystallographic studies.

Evidence for induced interactions in the anticooperative binding of nicotinamide adenine dinucleotide to sturgeon muscle glyceraldehyde-3-phosphate dehydrogenase

Evidence for induced interactions in the anticooperative binding of nicotinamide adenine dinucleotide to sturgeon muscle glyceraldehyde-3-phosphate dehydrogenase

Affinity Chromatography of Glyceraldehyde-3-phosphate Dehydrogenase. A Comparative Study of the Enzymes from Yeast and Sturgeon Muscle

Sturgeon glyceraldehyde-3-phosphate dehydrogenase has been strongly adsorbed on an 8-azo-linked NAD+-Sepharose derivative. Specific elution of the matrix-bound enzyme was obtained with relatively low concentrations of NAD+ (20–100 μM) in elution buffer. Recovery of adsorbed enzyme amounted to 90–95% when an hydrophilic 1,3-diaminopropan-2-ol spacer arm was used instead of the more conventional diaminohexyl derivative. A capacity of 0.8 mg of sturgeon enzyme per g of derivative was estimated either under batchwise conditions or by frontal chromatography. Under similar conditions, purified apoglyceraldehyde-3-phosphate dehydrogenase from yeast did not bind significantly to the same NAD+ derivative. Crude extracts of sturgeon muscle can be purified more than 15-fold upon chromatography on this derivative. Unlike the sturgeon enzyme, yeast glyceraldehyde-3-phosphate dehydrogenase was significantly adsorbed to an N6-(6-aminohexyl)-AMP derivative under chromatographic or batchwise conditions. At least 40 units (i.e. 0.2–0.3 mg) of yeast glyceraldehyde-3-phosphate dehydrogenase can be bound per g of matrix. An 8-fold purification of the enzyme can be obtained upon specific elution with NAD+. Relatively large amounts of yeast enzyme (i.e. up to 100 mg) can be partially purified by this one-step procedure. The distinct adsorption properties of yeast and sturgeon glyceraldehyde-3-phosphate dehydrogenases are discussed in the light of the known cooperative coenzyme binding properties of these enzymes. The relevance of affinity chromatography to the preparation of sizeable amounts of glyceral-dehyde-3-phosphate dehydrogenase is discussed and compared with conventional procedures. Possible interference of glyceraldehyde-3-phosphate dehydrogenase with the ‘general ligand’ binding of other dehydrogenase and kinases in crude extracts is discussed.

On the function of half-site reactivity: Intersubunit NAD +-dependent activation of acyl-glyceraldehyde 3-phosphate dehydrogenase reduction by NADH

Glyceraldehyde 3-phosphate dehydrogenase extracted from sturgeon muscle, exhibits half-site reactivity in its reaction with the pseudo-substrate β -(2-furyl) acryloyl phosphate ( Malhotra & Bernhard, 1968 ). The product is the difurylacryloyl thiol ester enzyme tetramer formed from the active site cysteinyl residue. The electronic spectrum of the furylacryloyl thiol ester linkage is perturbed upon binding of oxidized coenzyme (NAD + ) at the acyl site ( Malhotra & Bernhard, 1973 ). Likewise, we now report the perturbation in electronic spectrum of this furylacryloyl thiol ester upon interaction with NADH at the acyl site. The spectral perturbation accompanying NADH binding is large and in the opposite direction from that due to the binding of NAD + . The “color” of the chromophoretic acyl-enzyme linkage is a reflection of its chemical properties: furylacryloyl-enzyme-NAD complex is “red-shifter” (λ max =360 nm), and reacts uniquely with inorganic phosphate to yield the corresponding acyl phosphate. Furylacryloyl-enzyme-NADH complex is “blue-shifter” (λ max =330 nm) and is uniquely capable of reduction by NADH. Despite the presence of a ubiquitous NADH-induced color change, NADH reduction occurs only when NAD is bound to the remaining two non-acylated subunits. Hence, these results provide a direct example of allosteric regulation of enzyme function by ligand interaction at an adjacent subunit site. This site is known to be at least 17 A removed from the site of reduction ( Buehner et al. , 1974 ). Carboxymethylation of the two remaining (active) thiol groups of the diacyl-enzyme tetramer, a process which modifies the affinity for NAD at this site to only a small extent, does not affect the NAD-dependent activation of the acyl-NADH site for reduction. Qualitative transient kinetic evidence suggests that NAD binding to the non-acylated sites favors a slow conformational change leading to reducibility at acyl sites. The affinity of the various sites, acylated and non-acylated, for coenzyme ligands varies with the extent of acylation and the extent and nature of the ligand occupancies at adjacent subunits. Since the molar concentration of glyceraldehyde 3-phosphate dehydrogenase sites in muscle sarcoplasmic fluid is high (of the order of 1 pm), these results suggest that the “enzyme” functions as a regulatory warehouse for energy via acyl-enzyme-ATP interconversion and NADH/NAD redox potential regulation.

Relationship between structure and chemical reactivity in d-glyceraldehyde 3-phosphate dehydrogenase. Trinitrophenylation of the lysine residues in yeast, sturgeon and rabbit muscle enzyme

From the modification of the lysine residues of yeast and sturgeon muscle glyceraldehyde 3-phosphate dehydrogenase by 2,4,6-trinitrobenzene sulphonate the following results are obtained. 1. (a) In the absence of NAD + 1.1. (i) The trinitrophenylation follows a biphasic process. During the first phase two and four lysine residues are modified with a loss of 50% and 80% of the dehydrogenase activity in sturgeon muscle enzyme and yeast enzyme, respectively. 1.2. (ii) The modification of these more reactive lysine residues has very little effect on the K m of d-glyceraldehyde 3-phosphate, NAD + or arsenate; only V m is decreased; however, the esterase activity measured with p-nitrophenol acetate remains at about 85% of the initial activity. 1.3. (iii) The trinitrophenylation of these lysine residues in the yeast enzyme abolishes almost totally the S → N acetyl shift from cysteine 149 to lysine 183, whereas in the muscle apoenzymes it is partially prevented. 2. (b) In the presence of NAD + 2.1. (i) With the sturgeon muscle enzyme the trinitrophenylation is a first-order reaction and the loss of activity follows a first-order process, results which are similar to those obtained with the rabbit muscle enzyme (Foucault et al., 1974). Moreover, the rate of trinitrophenylation of lysine residues is identical to the rate of trinitrophenylation of the “slow” reacting lysine residues of apoenzyme, thus indicating that only the most reactive lysine residues are protected by the conformational change induced in the protein by the coenzyme. 2.2. (ii) With the yeast enzyme, trinitrophenylation of the holoenzyme still follows a bipliasic process; however, both rates are decreased as compared with the apoenzyme. The conformational changes induced by NAD + affect the reactivity of both classes of lysine residues. 3. (c) From the common effects of the trinitrophenylation of the most reactive lysine residues on the enzyme activity of the three dehydrogenases, as well as on the specific S → N acetyl transfer with acetylphosphate, it seems that the same lysine residues are being modified and that these might be either lysine 183 or lysine 191, which is located in the proximity. 4. (d) The difference in the number of lysine residues that are trinitrophenylated in the first phase of the chemical modification of the yeast and muscle enzymes is related either to the geometrical disposition of the subunits in the oligomeric structure or to the distance between the active centre regions which are face to face in the dimers (Buehner et al., 1974).

Differential effects of pyridoxal 5′-phosphate on lysine residues in rabbit and sturgeon muscle aldolases

Pyridoxal 5'-phosphate (PLP) in aqueous solutions can form a Schiff base complex with 14 and 16 lysine residues of rabbit and sturgeon muscle aldolases (EC 4.1.2.13), respectively. Although the mechanism of their interaction with PLP should be the same, these residues can be differentiated into three families on the basis of their inhibition constant Ki and rate constant k. The lysine residues of one of these families do not react with PLP in the presence of the substrates. Therefore, they are assumed to be part of the active center. In the sturgeon muscle aldolase, 3.7 substrate protected lysine residues are present. Rabbit aldolase, although tetrameric, contains only 2.8 substrate protected lysine residues. This suggests that one active center of this enzyme may be 'buried'. Structural studies showed the following sequence around the substrate protected lysine residues, in the rabbit aldolase: Gly-(Gly2, Val3)-Pyridoxyl Lys-Ile-Asp-Lys.

The binding of nicotinamide-adenine dimucleotide to glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

The binding of NAD+ to glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Bacillus stearothermophilus has been studied by measurement of protein fluorescence quenching. Slight negative co-operativity was observed in the binding of the third and fourth coenzyme molecules to the tetrameric enzyme. The first two coenzyme molecules were tightly bound. In this respect the enzyme resembles that from sturgeon muscle rather than that from yeast.

Purification and properties of the pyruvate kinase of sturgeon muscle

Pyruvate kinase was purified from sturgeon muscle in yeilds comparable with those obtained from the muscles of other species. In contrast with mammalian muscle pyruvate kinase the enzyme from sturgeon muscle gives a sigmoidal velocity curve with respect to phosphoenolpuruvate saturation, is activated by fructose 1.6-diphosphate, and is inhibited by bivalent copper ions. In these respects it is similar to the enzyme isolated from mammalian liver. The degree of interaction between phosphoenolpyruvate-binding sites is dependent on temperature.

Kinetic studies on oxidized nicotinamide--adenine dinucleotide-facilitated reactions of D-glyceraldehyde 3-phosphate dehydrogenase.

The kinetics of the acylation of d-glyceraldehyde 3-phosphate dehydrogenase from pig muscle by 1,3-diphosphoglycerate in the presence of NAD(+) has been analysed by using the relaxation temperature-jump method. At pH7.2 and 8 degrees C the rate of acylation of the NAD(+)-bound (or holo-) enzyme was 3.3x10(5)m(-1).s(-1) and the rate of phosphorolysis, the reverse reaction, was 7.5x10(3)m(-1).s(-1). After a temperature-jump perturbation the equilibrium of NAD(+) binding to the acyl-enzyme was re-established more rapidly than that of the acylation. The rate of phosphorolysis of the apoacylenzyme from sturgeon muscle and of aldehyde release from the d-glyceraldehyde 3-phosphate-apoenzyme complex were </=40m(-1).s(-1) and </=12s(-1) respectively at pH8.0 and 22 degrees C, which means that both processes are too slow to contribute significantly to the reaction pathway of the reversible NAD(+)-linked oxidative phosphorylation of d-glyceraldehyde 3-phosphate. Phosphorolysis of both acyl-apoenzyme and acyl-holoenzyme was first-order in P(i) up to 100mm-P(i) and more. PO(4) (3-) could be the reactive species of the phosphorolysis of the acyl-holoenzyme, in which case phosphorolysis is a diffusion-controlled reaction, although other kinetically indistinguishable rate equations for the reaction are possible.

On site heterogeneity in sturgeon muscle GPDH: a kinetic approach

The kinetics and stoichiometry of the reaction of sturgeon muscle glyceraldchyde-3-PO 4-dehydrogenase (GPDH) with the disulfide interchange reagent bis(2,2' dithio-bis(5-nitrobenzoate) (DTNB) has been studied in detail. The native enzyme, a tetramer of covalently identical subunits, reacts relatively rapidly with precisely four equivalents of reagent, although there are three cysteine residues per subunit (12 per tetratner). Reaction of these four cystcines leads to total catalytic inactivation; the extent of inactivation is proportional to the fractional reaction. The rate of reaction is dependent on the extent of bound NAD: reactivity being very much greater at unliganded sites. The reaction with apo-enzyme is fastest, bimolecular and monophasic. Over a wide range of NAD concentration, however, the reaction of enzyme with a large molar excess of reagent is precisely biphasic, and each individual kinetic experiment can be analytically described by two pseudo first-order (NAD concentration-dependent) rate constants and two unequal NAD concentration-insensitive amplitudes. The biphasicity in rate is quantitatively explainable on the basis of a C 2 symmetry for the tetrameric subunits with a tighter binding of NAD at two of the four sites, if high reactivity is exclusively dependent on the absence of bound NAD. The inequality in the two amplitudes, however, requires either a more complex or a more dynamic model. Arguments are presented for the appropriateness of a C 2 symmetry model in which intramolecular transconformational isomerization of tight and loose NAD binding sites is possible. The equilibrium constant for the isomerization is estimable from the macroscopic specific rates and amplitudes. This “flip-over” C 2 symmetry model is apropos to all situations of negative cooperativity in ligand binding to tetramers, as is discussed.

Preparation and active-site specific properties of sturgeon muscle glyceraldehyde-3-phosphate dehydrogenase

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPreparation and active-site specific properties of sturgeon muscle glyceraldehyde-3-phosphate dehydrogenaseFrancois Seydoux, Sidney Bernhard, Oswald Pfenninger, Mike Payne, and O. P. MalhotraCite this: Biochemistry 1973, 12, 21, 4290–4300Publication Date (Print):October 1, 1973Publication History Published online1 May 2002Published inissue 1 October 1973https://doi.org/10.1021/bi00745a038RIGHTS & PERMISSIONSArticle Views120Altmetric-Citations95LEARN 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 amino acid sequence of the N-terminal region of aldolase.

The amino acid sequence of the N-terminal 39 residues of sturgeon muscle aldolase has been determined by a combination of automated and manual sequencing procedures. In addition, the sequence of the N-terminal 19 residues of rabbit muscle aldolase has been determined using a sequenator. It was apparent from these studies that aldolase primary structure is highly conserved in this region.

Amino-acid sequence homology in the muscle aldolases from sturgeons of different species.

STUDIES on the primary structure of aldolases isolated from ox, pig and rabbit muscle show that the amino-acid sequence of fructose 1,6-diphosphate aldolase [EC 4.1.2.13] has been highly conserved throughout mammalian evolution1. But comparison of the primary structure of the enzyme from these species with that from the muscle of a single North Sea sturgeon, presumably Acipenser sturio, indicated that although the proteins were homologous, a number of amino-acid replacements occurred between sturgeon aldolase and the aldolases of the phylogenetically distant mammalian species1. As a knowledge of the nature and number of amino-acid replacements between homologous proteins caft provide information both about the functional role of individual residues and about evolution, further comparative studies of rabbit and sturgeon aldolases were undertaken and an account of the sequence homology around the active-site-lysine residue of aldolases from rabbit muscle, rabbit liver and the muscle of the river sturgeon of Eastern Canada, Acipenser fulvescens, has been given2,3.

The number, location, and reactivity of the cysteine residues of sturgeon muscle aldolase.

Sturgeon muscle aldolase contains six cysteine residues per subunit. These residues appear to occur in homologous positions to six of the eight cysteine residues of rabbit muscle aldolase. Three of the six residues can react with either iodoacetic acid or 5,5′-dithiobis-(2-nitrobenzoic acid) in the absence of denaturing agents. Reaction of three residues with iodoacetic acid inactivates the enzyme. The presence of substrate protects one of these residues and in this case no activity loss is observed. However, the results indicate that the effects on activity are due to the addition of the modifying group rather than to a loss of sulfhydryl groups. No residue corresponding to the previously demonstrated substrate-protected cysteine of rabbit aldolase was located in the sturgeon enzyme, which demonstrated that this residue was not essential to aldolase activity. It therefore appears unlikely that cysteine residues have a direct or an auxiliary catalytic role in aldolase activity.

The carboxy-terminal region of sturgeon muscle aldolase.

The modification of the carboxy-terminal of sturgeon muscle aldolase with carboxypeptidase produces similar kinetic effects to those observed on modification of the rabbit muscle enzyme. The nature and molar ratios of amino acids released indicate that differences between the two enzymes occur in the carboxy-terminal region.

Amino acid sequence homology in the active site of rabbit and sturgeon muscle aldolases

Amino acid sequence homology in the active site of rabbit and sturgeon muscle aldolases

A comparative study of the structure of muscle fructose 1,6-diphosphate aldolases.

1 The primary structures of fructose diphosphate aldolases from the skeletal muscle of rabbit, pig, ox and sturgeon have been compared by means of zone electrophoresis, amino acid and N-terminal analysis, peptide mapping and cyanogen bromide cleavage. 2 The S-carboxymethylated mammalian enzymes give two bands on polyacrylamide gel electrophoresis, under conditions where the S-carboxymethylated sturgeon enzyme gives only one. The two bands usually stain unequally with the rabbit enzyme, whereas with the ox and pig enzymes a more equal intensity is observed. 3 The mammalian enzymes are highly homologous by the criteria used, and it seems likely that the sturgeon enzyme will be fairly closely alike. In this degree of homology, the aldolases resemble the glyceraldehyde 3-phosphate dehydrogenases from various sources, suggesting that such homology may be a common feature of glycolytic enzymes. 4 Despite the occurrence of two bands in gel electrophoresis of the mammalian enzymes, it is clear that there cannot be substantial sequence differences between the subunit peptide chains of a given aldolase. Such differences, if they exist, must be relatively few in number. Other explanations, such as chemical modification of some of the chains, may account for the observed electrophoretic behaviour in the mammalian enzymes. 5 The ox enzyme contains one additonal cysteine residue per subunit compared with the other mammalian enzymes. This change has been located in the primary structure and since the cysteine residue is reactive in the native ox enzyme, it may provide a useful site for attachment of heavy atoms in X-ray crystallographic studies.