Rabbit Muscle Enzyme Research Articles

The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms.

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues’ contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation.

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium.

New insights on the mechanism of the K(+-) independent activity of crenarchaeota pyruvate kinases.

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent catalysis.

Characterization of enzymatically synthesized amylopectin analogs via asymmetrical flow field flow fractionation

Asymmetrical flow field flow fractionation (AF4), when coupled with multi-angle laser light scattering (MALLS), is a very powerful technique for determination of the macromolecular structure of high molar mass (branched) polysaccharides. AF4 is a size fractionation technique just as size exclusion chromatography (SEC), nevertheless can overcome some crucial problems found in SEC analysis especially in starch like structures. This paper describes a detailed investigation of the macromolecular structure of two groups of well-defined synthetic amylopectin analogs – synthesized via an in vitro enzyme-catalyzed reaction using the enzymes phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme (Dg GBE). Size, molar mass distributions and structural data were studied by AF4 coupled with online quasi-elastic light scattering (QELS) and multi-angle light scattering (MALLS).

Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca²⁺-ATPase.

Infrared spectroscopy was used to characterise recombinant sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). In the amide I region, its spectrum differed from that of Ca(2+)-ATPase prepared from rabbit fast twitch muscle below 1650 cm(-1). A band at 1642 cm(-1) is reduced in the spectrum of the recombinant protein and a band at 1631 cm(-1) is more prominent. By comparison of amide I band areas with the known secondary structure content of the protein, we assigned the 1642 cm(-1) band to β-sheet structure. Further investigation revealed that the 1642 cm(-1) band decreased and the 1631 cm(-1) band increased upon storage at room temperature and upon repeated washing of a protein film with water. Also protein aggregates obtained after solubilisation of the rabbit muscle enzyme showed a prominent band at 1631 cm(-1), whereas the spectrum of solubilised ATPase resembled that of the membrane bound protein. The spectral position of the 1631 cm(-1) band is similar to that of a band observed for inclusion bodies of other proteins. The findings show that the absence of the 1642 cm(-1) band and the presence of a prominent band at 1631 cm(-1) indicate protein aggregation and can be used as a quality marker for the optimisation of recombinant protein production. We conclude that recombinant production of SERCA1a, storage at room temperature, repeated washing and aggregation after solubilisation all modify existing β-sheets in the cytosolic domains so that they become similar to those found in inclusion bodies of other proteins.

Size Exclusion Chromatography with Multi Detection in Combination with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry as a Tool for Unraveling the Mechanism of the Enzymatic Polymerization of Polysaccharides

Determination of the size distributions of natural polysaccharides is a challenging task. More advantageous for characterization are well-defined synthetic (hyper)-branched polymers. In this study we concentrated on synthetic amylopectin analogues in order to obtain and compare all available data for different distributions and size dependence of molecular weights. Two groups of well-defined synthetic branched polysaccharides were synthesized via an in vitro enzyme-catalyzed reaction using the enzyme phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme. Synthetic polymers had a tunable degree of branching (2%-13% determined via (1)H NMR) and a tunable degree of polymerization (30-350 determined indirectly via UV spectrometry). The systems used for separation and characterization of branched polysaccharides were SEC-DMSO/LiBr and multi detection (refractive index detector, viscosity detector, and multi angle light scattering detector) and SEC-water/0.02% NaN(3); and SEC-50 mM NaNO(3)/0.02% NaN(3) and multi detection. Additionally the side chain length distribution of enzymatically debranched polysaccharides was investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. With this combination of characterization techniques, we were able not only to characterize the amylopectin analogues but also to solve parts of the molecular mechanism of their enzymatic polymerization. Moreover our materials showed potential to be standards in the field of natural polysaccharides characterization.

Synthesis of branched polysaccharides with tunable degree of branching

An in vitro enzyme-catalyzed tandem reaction using the enzymes phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) to obtain branched polyglucans with tunable degree of branching (2% ÷ 13%) is presented. The tunable degree of branching is obtained by varying the reaction conditions such as pH value, the choice of reducing agent and its concentration and reaction time. Linear amylose is formed by the phosphorylase-catalyzed propagation of glucose-1-phosphate while Dg GBE introduces branching points on the α-(1→6) position by relocating short oligosaccharide chains. Our results show that the best way to obtain different degrees of branching with this set of enzymes is by regulation of the reaction time.

Discovering benzamide derivatives as glycogen phosphorylase inhibitors and their binding site at the enzyme

A series of novel benzamide derivatives was designed, synthesized, and their inhibitory activities against glycogen phosphorylase (GP) in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate were evaluated. The structure-activity relationships (SAR) of these compounds are also presented. Within this series of compounds, 4m is the most potent GPa inhibitor (IC 50 = 2.68 μM), which is nearly 100 times more potent than the initial compound 1. Analysis of mapping between pharmacophores of different binding sites and each compound demonstrated that these benzamide derivatives bind at the dimer interface of the rabbit muscle enzyme, and possible docking modes of compound 4m were explored by molecular docking simulation.

Novel thienopyrrole glycogen phosphorylase inhibitors: Synthesis, in vitro SAR and crystallographic studies

Two series of novel thienopyrrole inhibitors of recombinant human liver glycogen phosphorylase a (GPa) which are effective in reducing glucose output from rat hepatocytes are described. Representative compounds have been shown to bind at the dimer interface site of the rabbit muscle enzyme by X-ray crystallography.

Glycolytic Enzyme Interactions with Yeast and Skeletal Muscle F-Actin

Interaction of glycolytic enzymes with F-actin is suggested to be a mechanism for compartmentation of the glycolytic pathway. Earlier work demonstrates that muscle F-actin strongly binds glycolytic enzymes, allowing for the general conclusion that “actin binds enzymes”, which may be a generalized phenomenon. By taking actin from a lower form, such as yeast, which is more deviant from muscle actin than other higher animal forms, the generality of glycolytic enzyme interactions with actin and the cytoskeleton can be tested and compared with higher eukaryotes, e.g., rabbit muscle. Cosedimentation of rabbit skeletal muscle and yeast F-actin with muscle fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by Scatchard analysis revealed a biphasic binding, indicating high- and low-affinity domains. Muscle aldolase and GAPDH showed low-affinity for binding yeast F-actin, presumably because of fewer acidic residues at the N-terminus of yeast actin; this difference in affinity is also seen in Brownian dynamics computer simulations. Yeast GAPDH and aldolase showed low-affinity binding to yeast actin, which suggests that actin-glycolytic enzyme interactions may also occur in yeast although with lower affinity than in higher eukaryotes. The cosedimentation results were supported by viscometry results that revealed significant cross-linking at lower concentrations of rabbit muscle enzymes than yeast enzymes. Brownian dynamics simulations of yeast and muscle aldolase and GAPDH with yeast and muscle actin compared the relative association free energy. Yeast aldolase did not specifically bind to either yeast or muscle actin. Yeast GAPDH did bind to yeast actin although with a much lower affinity than when binding muscle actin. The binding of yeast enzymes to yeast actin was much less site specific and showed much lower affinities than in the case with muscle enzymes and muscle actin.

Regulation of Glycogen Synthase: IDENTIFICATION OF RESIDUES INVOLVED IN REGULATION BY THE ALLOSTERIC LIGAND GLUCOSE-6-P AND BY PHOSPHORYLATION

Regulation of Glycogen Synthase: IDENTIFICATION OF RESIDUES INVOLVED IN REGULATION BY THE ALLOSTERIC LIGAND GLUCOSE-6-P AND BY PHOSPHORYLATION

A Recombinant Form of the Catalytic Subunit of Phosphorylase Kinase That Is Soluble, Monomeric, and Includes Key C-Terminal Residues

Residues 302–326 of the catalytic (γ) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKγ was expressed using Escherichia coli. This yielded two stable, high-activity PhKγ forms (35 and 42 kDa by SDS–PAGE) that were smaller than an authentic sample of rabbit muscle PhKγ (45 kDa by SDS–PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKγ residues 1–362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact γ subunit. This implied that the lack of 25 PhKγ C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKγ that consisted of residues 1–300, denoted γ1–300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Novel Aspects of Tetramer Assembly and N-terminal Domain Structure and Function Are Revealed by Recombinant Expression of Human AMP Deaminase Isoforms

AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4 degreesC. Expression of N-truncated cDNAs (DeltaL96AMPD1 and DeltaM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher Km(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4 degreesC. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.

The Monovalent Cation Requirement of Rabbit Muscle Pyruvate Kinase Is Eliminated by Substitution of Lysine for Glutamate 117

The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+[T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994)Biochemistry33, 6301–6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases fromCorynebacterium glutamicumandEscherichia coli,which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated ϵ-amino group of Lys 117 in these bacterial enzymes may provide an “internal monovalent cation.” Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is >200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated ϵ-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

The kinetic and equilibrium isotope effects on the fructose-1, 6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme. The natural 13C abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3-phosphoglycerate, respectively, and chemical degradation. The carbon isotope effects were determined comparing the 13C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13(Vmax/Km) on C-3 was 1.016 +/- 0.007 and 0.997 +/- 0.009 on position C-4, and the equilibrium isotope effects K12/K13 on these positions were 1.0036 +/- 0.0002 and 1.0049 +/- 0.0001. The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative 13C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.

Primary Structure of Human Liver Glycogen Synthase Deduced by cDNA Cloning

The cDNA for human liver glycogen synthase was isolated by screening a human liver cDNA library constructed in λgt11. The full cDNA was 2912 bp in length. It coded for a protein of 703 amino acid residues with a molecular mass of 80.9 kDa. The number of amino acids was identical to and the deduced amino acid sequence homology was 92% that of the rat liver enzyme. The human and rat liver glycogen synthases are truncated by 34 amino acids compared to the human muscle enzyme, and by 32 amino acids compared to the rabbit muscle enzyme. The amino acid similarity between human liver and human muscle glycogen synthase was only 69%. It was least similar in the N and C terminal regions of the molecule. Two highly conserved regions are present in all published amino acid sequences for glycogen synthase, including those of the two yeast enzymes. These regions include the amino acid sequences from 201 to 400 and 501 to 600. This high conservation suggests that the catalytic site and the glucose-6-P and nucleotide allosteric sites are included in these regions.

Conformational changes in proteins induced by dynamic associations. A tryptophan phosphorescence study

Random collisions between macromolecules lead to dynamic associations (lengthy encounters) that in principle could affect their conformation and, in the case of enzymes, their binding and catalytic properties. Exploiting the unique sensitivity of the phosphorescence lifetime, tau, of Trp to the internal flexibility of globular proteins we probed the perturbations induced in the structure of the coenzyme-binding domain of alcohol dehydrogenase (LADH) and glyceraldehyde-3-phosphate dehydrogenase (GraPDH) by the presence in solution of other dehydrogenases and of functionally unrelated proteins. With Trp314 of LADH, the results emphasize that while tau is not affected by the concentration of LADH itself, the addition of micromolar quantities of other proteins causes a distinct reduction in it. From the linear increase of 1/tau with protein concentration one obtains values for the apparent second-order Stern-Volmer rate constant that range between 2-200 x 10(3) M-1 s-1, decreasing 2-3-fold when ternary complexes of LADH with NADH or NAD+ and inhibitors are involved. Similar effects were observed with Trp310 of GraPDH except that with sorbitol dehydrogenase as perturbant the increase of 1/tau is hyperbolic and governed by an apparent dissociation constant of about 1 microM. Finally, glycerol-3-phosphate dehydrogenase, the strongest perturber of both LADH and GraPDH, has either no effect on lactic dehydrogenase from pig heart or induces a moderate lengthening of the triplet lifetime of the rabbit muscle enzyme. Because Stern-Volmer behavior is typical also of diffusion-mediated quenching reactions, a parallel investigation with cysteine, cystine and N-acetyl-tryptophanamide demonstrated that among potential, protein-associated, quenching moieties namely, -SH, -S-S- and indole groups, only the latter has rate constants approaching the magnitude of protein perturbants. Since considerable evidence rules out the predominance of such quenching reactions, these findings confirm a subtle form of communication between protein molecules in solution. The lack of specificity and the similar effects between dehydrogenases with right and wrong stereospecificity for direct coenzyme transfer suggests that the perturbations monitored are unrelated to this function.

The catalytic activity of adenosine monophosphate deaminase in murine thymus as revealed by Transmission Electron Microscopy

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The caloro-analog of AMP, i.e., 6 Chloropurine riboside 5′ monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which liberates Cl− which is precipitated with added Ag+, and, after exposure to light electron sense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K+ (as the acetate), 3mM ATP and 10μM pentostatin deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this Enzyme. Because many cells contain ecto 5′-nucleotidase activity, pentostatin was added to prevent manifestation of denosine deaminase activity of dechlorination of any nucleoside that might be formed. Any Cl− formed by adenosine deaminase would be precipitated with the added Ag+ and thus result in Ago deposits which would be indistinguishable from the Ago deposited by AMP deaminase.

The catalytic activity of adenosine monophosphate deaminase in murine duodenum as revealed by transmission electron microscopy

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The chloro-analog of AMP, i.e., 6 Chloropurine riboside 5' monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which liberates Cl- which is precipitated with added Ag+, and, after exposure to light electron dense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K+ (as the acetate), 3mM ATP and 10¼M pentostathi (deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this enzyme. Because many cells have ecto 5'-nucleotidase activity, pentostathi was added to prevent manifestation of adenosine deaminase activity of dechlorination of any nucleoside that might be formed.

Phosphofructokinase from white muscle of the rainbow trout, Oncorhynchus mykiss: purification and properties

Phosphofructokinase was purified and characterized from the white skeletal muscle of rainbow trout Oncorhynchus mykiss. Purification involved three steps: ion-exchange chromatography on hydroxyapatite and affinity chromatography on phosphocellulose and ATP-agarose. A final specific activity of 75 units per mg of protein at 22°C and pH 7.2 with 40% recovery was obtained. The purified enzyme gave a single band on SDS-PAGE with a subunit molecular mass of 76.5±0.6 kDa . Based on gel filtration analysis, the active form of the enzyme was found to be composed of six identical subunits. A high isoelectric point (7.1) was found for this enzyme. Arrhenius plots of the enzyme activity showed a sharp transition at 15–16°C. The pH optimum of the enzyme was 8.0–8.5 at physiological level of ATP and positive modulators shifted the optimum to lower pH values. Amino-acid analysis revealed a lower content of the aromatic residues Phe, Tyr and Trp and higher level of Ser residue than in the rabbit muscle enzyme.