Human Erythrocyte Phosphofructokinase Research Articles

The Enzyme Activity Allosteric Regulation Model Based on the Composite Nature of Catalytic and Regulatory Sites Concept

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure. The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.

Rapid purification of human erythrocyte phosphofructokinase

Rapid purification of human erythrocyte phosphofructokinase

Influence of free Mg 2+ on the kinetics of human erythrocyte phosphofructokinase

The influence of Mg2+ on the reaction catalyzed by human erythrocyte phosphofructokinase has been investigated using kinetic methods. The catalytic activity of PFK is dependent upon the presence of Mg2+ which constitutes with ATP the true Mg-ATP2- substrate. Free Mg2+ has no influence on the affinity of the enzyme for Mg-ATP2- substrate. Erythrocyte PFK is more inhibited by ATP4- and uncomplexed citrate than it is by Mg-ATP2- and Mg-citrate. Free Mg2+ relieves the MgATP2- and Mg-citrate inhibition under conditions where free ATP4-is negligible. We can assume that uncomplexed Mg2+ acts as positive effector by direct binding to the enzyme. These results emphasize the role of Mg2+ in the regulation of PFK activity in the erythrocyte.

The molecular mechanism of the inherited phosphofructokinase deficiency associated with hemolysis and myopathy

Normal human erythrocyte phosphofructokinase (ATP:c D-fructose-6, P-1- phosphotransferase, EC 2.7.1.11; PFK) has recently been shown to consist of a heterogeneous mixture of five tetrameric isozymes: M4, M3L, M2L2, ML3, and L4 (M, muscle type; L, liver type). In the light of these findings, we have investigated the molecular basis of the inherited erythrocyte PFK deficiency associated with myopathy and hemolysis (Tarui disease). The propositus, a 31-yr-old male, suffered from muscle weakness and myoglobinuria on exertion. He showed mild erythrocytosis despite laboratory evidence of hemolysis. In his erythrocytes a metabolic crossover point was found at the level of PFK; 2,3-diphosphoglycerate (2,3-DPG) was also significantly reduced. The PFK from the patient's erythrocytes consisted exclusively of the L4 isozyme, and there was a complete absence of the other four. The leukocyte and platelet PFKs from the patient showed normal activities, chromatographic profiles, and precipitation with anti-M4 antibody. These studies provide direct evidence that in Tarui disease the M-type subunits are absent; but the liver- and platelet-type subunits of PFK are unaffected. The paradox of mild erythrocytosis despite hemolysis reflects the decreased production of 2,3-DPG.

The Molecular Mechanism of the Inherited Phosphofructokinase Deficiency Associated With Hemolysis and Myopathy

Normal human erythrocyte phosphofructokinase (ATP: D-fructose-6, P-1-phosphotransferase, EC 2.7.1.11; PFK) has recently been shown to consist of a heterogeneous mixture of five tetrameric isozymes: M4, M3L, M2L2, ML3, and L4 (M, muscle type; L, liver type). In the light of these findings, we have investigated the molecular basis of the inherited erythrocyte PFK deficiency associated with myopathy and hemolysis (Tarui disease). The propositus, a 31-yr-old male, suffered from muscle weakness and myoglobinuria on exertion. He showed mild erythrocytosis despite laboratory evidence of hemolysis. In his erythrocytes a metabolic crossover point was found at the level of PFK; 2,3-diphosphoglycer-ate (2,3-DPG) was also significantly reduced. The PFK from the patient's erythrocytes consisted exclusively of the L4 isozyme, and there was a complete absence of the other four. The leukocyte and platelet PFKs from the patient showed normal activities, chromatographic profiles, and precipitation with anti-M4 antibody. These studies provide direct evidence that in Tarui disease the M-type subunits are absent; but the liver- and platelet-type subunits of PFK are unaffected. The paradox of mild erythrocytosis despite hemolysis reflects the decreased production of 2,3-DPG.

Nonidentical subunits of human erythrocyte phosphofructokinase.

Human erythrocyte and muscle phosphofructokinase (PFK) were purified completely by improved procedures. SDS-acrylamide gel electrophoresis in a discontinuous buffer system revealed two subunits (R and M) of erythrocyte PFK, the slower one (M) corresponding to the single subunit of muscle PFK. The staining intensity ratio R:M of the two bands of erythrocyte PFK was 2:1 or less. This suggests that native erythrocyte PFK contains multiple isoenzymes with different proportions of R and M, some being lost during purification. Nevertheless, isoelectric focusing showed single peaks of erythrocyte PFK (pI 5.0) and muscle PFK (pI 6.6), perhaps because of aggregation of erythrocyte PFK isoenzymes. Erythrocyte PFK from a patient with muscle PFK deficiency had a pI of 4.6 and could not be precipitated by antiserum against muscle PFK, findings compatible with the putative structure R4.

Activation of human erythrocyte pyruvate kinase by hexose-bisphosphates

Summary Human erythrocyte pyruvate kinase (E.C. 2.7.1.40, ATP-pyruvate phosphotransferase) is activated by hexose-1,6-bisphosphates in the order: fructose- > mannose- > glucose-bisphosphate. These activators promote substantial reductions (7 to 10 fold) in the K M for the substrate phosphoenolpyruvate but do not effect the maximum velocity. The ability of mannose- and glucose-1,6-bisphosphate to function as activators of both human erythrocyte phosphofructokinase (Rose, I.A. and Warms, J.V.B. (1974), Biochem. Biophys. Acta 156, 231–239) and pyruvate kinase may account for the presence of these metabolites in red cells at relatively high levels. Fructose-1,6-bisphosphate also induces slight increases in the K M values for the active substrate, Mg·ADP, at sub-saturating levels of phosphoenolpyruvate but is without effect at saturating levels of phosphoenolpyruvate. A possible role for these activators in maintaining a balance in the flux through the pyruvate kinase reaction is proposed.

Studies on structure of human erythrocyte phosphofructokinase.

Studies on structure of human erythrocyte phosphofructokinase.

Self-association of human erythrocyte phosphofructokinase. Kinetic behaviour in dependence on enzyme concentration and mode of association.

The kinetic behaviour of human erythrocyte phosphofructokinase has been analyzed over a relative wide range of enzyme concentration (0.01 -- 1.7 mug/ml). The kinetic cooperativity which becomes apparent when the enzymic reaction rate is plotted versus the fructose 6-phosphate concentration decreases with increasing enzyme concentration. Simultaneously, a decrease of the half-saturation concentration for fructose 6-phosphate [S]0.5 is observed. Maximum velocity passes through a maximum at increasing enzyme concentrations. Sets of curves representing specific enzymic activity of phosphofructokinase versus enzyme concentration obtained at various fixed concentrations of fructose 6-phosphate and ATP are analyzed. The shapes of these curves are interpreted in terms of an association model of human erythrocyte phosphofructokinase, in which an inactive dimer (Mr 190000) and active multimers of the dimeric form are involved. The conclusion is drawn that the sigmoidal shape of the plots of the enzymic reaction rate versus fructose 6-phosphate concentration is partially caused by a displacement of the equilibrium between different states of association of phosphofructokinase to multimers by this substrate. On the other hand, the inhibition of the enzyme by high concentrations of ATP may be partially caused by a shift of this equilibrium to the state of the inactive dimer.

Association-dissociation behavior of erythrocyte phosphofructokinase and tumor pyruvate kinase

The association-dissociation behavior of human erythrocyte phosphofructokinase and of Ehrlich ascites tumor cell pyruvate kinase has been studied. The kinetic properties of erythrocyte phosphofructokinase depend on its state of association. With increasing enzyme concentrations (1) the specific activity passes through a maximum, (2) the Hill-coefficient for fructose 6-phosphate decreases, (3) the affinity of the enzyme to fructose 6-phosphate increases, and (4) the ATP inhibition decreases. The association of human erythrocyte phosphofructokinase in dependence on the protein concentration has been followed by means of frontal gel chromatography. A cooperative association model describing satisfactorily the kinetic behavior of this type of phosphofructokinase is suggested. Ehrlich ascites tumor cell pyruvate kinase resembles in its electrophoretic and kinetic properties the M 2-type of the enzyme in mammalian tissues. It is inactivated by l-alanine at low temperatures. Inactivation may be reversed by rewarming of the enzyme or by fructose 1,6-diphosphate or by phosphoenolpyruvate, respectively. The inactivation-reactivation cycle is related to a dimer-tetramer transition of the enzyme. The dimer appears to be subactive, while the tetramer is supposed to be the enzymatic full active molecular species. Owing to the slow rate of the dimer-tetramer transition this type of tumor pyruvate kinase is regarded to react hysteretically. A theoretical three-state model is proposed for a qualitative and semiquantitative interpretation of the complex kinetic behavior of ascites tumor cell pyruvate kinase, involving one tetrameric and two kinetic different dimeric states of the enzyme.

The Molecular Basis of Muscle Phosphofructokinase Deficiency

Experiments were designed to test the hypothesis that human erythrocyte phosphofructokinase (PFK) is composed of nonidentical subunits (M and R) while muscle PFK is composed of M subunits only. When erythrocyte PFK was dissociated into inactive subunits at pH 5.3, the R subunit could be precipitated by a specific antibody, and the reassociated enzyme at pH 8.0 had the chromatographic and immunologic characteristics of muscle PFK. When dissociated erythrocyte PFK was treated with antiserum against muscle PFK and reassociated, the resultant enzyme no longer contained the M-type antigen. The chromatographic methods did not distinguish between native erythrocyte PFK and PFK from which the subunits had been removed. The results support the original hypothesis and provide a basis for explaining muscle PFK deficiency as a genetic lack of the M subunit.

Etude de quelques propriétés de la phosphofructokinase érythrocytaire de l'homme normal

Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation. At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg2+. ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules. The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it. These ligands had no effect on the maximum velocity of the reaction except in the case of ADP. Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins. With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase. Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %. Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG. The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group. La phosphofructokinase d'érythrocyte humain a été purifiée environ 150 fois par adsorption sur DEAE cellulose et précipitation par le sulfate d'ammonium. A pH 7,5 l'enzyme révèle une cinétique allostérique vis-à-vis de l'ATP, du F6P et du Mg2+. L'ATP à forte concentration joue le rôle d'inhibiteur, et l'ADP, le 5'AMP, le 3′5′AMP jouent le rôle d'activateurs. Ces effecteurs semblent diminuer les interactions homotropes entre les molécules de F6P. Les activateurs augmentent l'affinité de la phosphofructokinase pour le susbtrat F6P l'inhibiteur la diminue. Ces ligands n'ont pas d'effet sur la vitesse maximale de la réaction sauf dans le cas de l'ADP. Les interactions entre les molécules de substrat et de ligands effecteurs sont considérées dans les termes du modèle défini par Monod - Changeux - Wyman pour les protéines oligomériques. Avec le GTP et l'ITP, aucune inhibition n'est observée. A concentration saturante de GTP l'ATP inhibe encore la phosphofructokinase. Le 3′5′ AMP et le F6P augmentent la concentration d'ATP nécessaire pour produire 50 % d'inhibition. Le citrate, tout comme l'ATP inhibe la phosphofructokinase en se fixant probablement au même site allostérique. La phosphofructokinase d'érythrocyte est inhibée par le 2–3 DPG. L'étude de la relation log V max = f (pH) suggère que le centre actif contient au moins un groupement imidazol et un groupement sulfhydryl.

Studies on the association behaviour of human-erythrocyte phosphofructokinase.

By applying analytical gel chromatography technique the association behaviour of human erythrocyte phosphofructokinase has been studied. The molecular weight distribution of the polymerized forms of this enzyme covers a range of 380000 to about six millions. The degree of polymerization has been found to depend on the protein concentration, on the presence of various substrates and effectors and on the pH‐value. ATP favours the aggregation of the enzyme with increasing pH, whereas in presence of fructose 6‐phosphate or fructose 1,6‐bisphosphate respectively the degree of association appears to be independent of the pH value between pH 6 and 9. In the microgram range the specific activity of the enzyme decreases with an increase of the enzyme concentration.

Cooperativity in human erythrocyte phosphofructokinase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCooperativity in human erythrocyte phosphofructokinaseLilian M. Y. Lee, R. M. Krupka, and R. A. CookCite this: Biochemistry 1973, 12, 18, 3503–3508Publication Date (Print):August 1, 1973Publication History Published online1 May 2002Published inissue 1 August 1973https://doi.org/10.1021/bi00742a024RIGHTS & PERMISSIONSArticle Views58Altmetric-Citations13LEARN 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 (693 KB) Get e-Alerts Get e-Alerts

Human erythrocyte phosphofructokinase: Its Purification and some properties

1. 1. Human erythrocyte phosphofructokinase (ATP: d-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified 15 000-fold by (NH 4) 2SO 4 precipitation, heating and column chromatography on Sepharose 6-B. The resulting enzyme preparation had a specific activity of 60 μmoles Fru-1,6- P 2 formed per min per mg protein at 25 °C. 2. 2. With cellulose-acetate electrophoresis only one band was observed after detection of the enzyme activity with the fluorescent technique. 3. 3. Citrate and 2,3-diphosphoglycerate do not inhibit. The inhibition by ATP is pH dependent. Cyclic AMP is able to reverse the inhibition by ATP to some extent. 4. 4. With GTP, ITP and UTP no inhibition is observed. At saturating concentrations of GTP, ATP still inhibits phosphofructokinase. 5. 5. The variation of the activity of phosphofructokinase at various ATP and Fru-6- P concentrations was studied. In the reaction mechanism a ternary complex is involved.

Purification of human erythrocyte phosphofructokinase

Phosphofmctokinase* has been isolated from several mammalian tissues in highly purified or even crystallized forms [l-6] . Human PFK received greater attention when a new human disease was detected, which could be characterized by a deficiency of muscle PFK and a decrease of the enzyme in red blood cells to about 50% of normal. These findings led to comparative studies of various properties of both enzymes, which revealed characteristic differences between them [7-91 . In these studies only partially purified erythrocyte PFK with a specific activity of 12wasused [7]. Recently, we described the occurrence of various oligomeric forms of human erythrocyte PFK, and determined the molecular weights of their SDS cleavage products [lo] . In this paper the procedure for getting a highly purified, stable and homogeneous human erythrocyte PFK preparation with a specific activity of about 130 is described. Some special properties of the enzyme are also presented.

Evidence for different oligomeric forms of human erythrocyte phosphofructokinase

Phosphofructokinases from mammalian tissues occur in different association states under a variety of experimental conditions [l-6]. These states depend on protein concentration and were found to be influenced by substrates and effecters as well as by changing pH values and ionic strengths. Layzer and coworkers [7,8] described some properties of partially purified erythrocyte phosphofructokinase including a concentration-dependent sedimentation behaviour of the enzyme using density gradient centrifugation. The present paper reports the existence of a series of oligomeric forms of human erythrocyte phosphofructokinase in highly purified enzyme preparations. These are correlated with a product of splitting of the enzyme having a molecular weight of 104,000 which could be characterized by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis.