Biomedical subjects
Publications and source records attributed to S Beeckmans.
One of the authors was omitted in the published version of the paper by Lisgarten et al. (1999) Acta Cryst. D55, 1903-1905. The full author list is given above.
Proteins and enzymes are now generally thought to be organized within the cell to form clusters in a dynamic and versatile way, and heterologous protein-protein interactions are believed to be involved in virtually all cellular events. Therefore we need appropriate tools to detect and study such interactions. Chromatographic techniques prove to be well suited for this kind of investigation. Real complexes formed between proteins can be studied by classic gel filtration. When enzymes are studied, active enzyme gel chromatography is a useful alternative. A variant of classic gel filtration is gel filtration equilibrium analysis, which is similar to equilibrium dialysis. When the association formed is only dynamic and equilibrates very rapidly, either the Hummel-Dryer method of equilibrium gel filtration or large-zone equilibrium filtration sometimes allows the interactions to be analyzed, both qualitatively and quantitatively. Very often, however, interactions between enzymes and proteins can only be evidenced in vitro in media that mimic the intracellular situation. Immobilized proteins are excellent tools for this type of research. Several examples are indeed known where the immobilization of an enzyme on a solid support does not affect its real properties, but rather changes its environment in such a way that the diffusion becomes limiting. Affinity chromatography using immobilized proteins allows the analysis of heterologous protein-protein interactions, both qualitatively and quantitatively. A useful alternative appears to be affinity electrophoresis. The latter technique, however, is exclusively qualitative. All these techniques are described and illustrated with examples taken from the literature.
Crystals of Helix pomatia agglutinin (HPA) have been grown by the hanging-drop technique using polyethylene glycol as the precipitant at 293 K. Over a period of one to two weeks the crystals grew to maximum dimensions of 0.10 x 0.05 x 0.02 mm. The crystals belong to space group P6(3)22, with unit-cell dimensions a = b = 63.3, c = 105. 2 A and Z = 12 identical monomers of M(r) = 13 kDa, aggregating into two 78 kDa hexameric protein molecules per unit cell, each with symmetry 32 (D(3)). The diffraction pattern extends to 3.6 A at 293 K.
A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).
The role of Mg2+ in the structure and activity of maize isocitrate lyase has been studied by CD, limited proteolysis, protection by ligands against inactivation, and activity measurements at various metal concentrations. From CD and trypsinolysis experiments, the existence of high-affinity binding sites for Mg2+ was demonstrated, and a KdME of 200 microM was determined. Both free enzyme (E) and enzyme molecules with high-affinity sites occupied (ME) are catalytically competent, the former showing 40% of the activity of the latter. Mg2+ thus acts as a non-essential activator. A second Mg2+-binding site with a KdMEM of 6 mM was revealed from protection experiments by increasing Mg2+ concentrations against inactivation. From activity measurements at different Mg2+ concentrations, the affinity of the enzyme for the Mg2+-isocitrate complex (MI) was determined to be KdE(MI) = 9 microM. Maize isocitrate lyase was shown to display hysteretic behaviour. Filling the low-affinity binding sites with Mg2+ induces a conformational change in the high-affinity binding sites resulting in an even higher affinity for Mg2+ (KdME* = 40 microM). On lowering the Mg2+ concentration again, the enzyme only responds slowly: the time needed for all enzyme molecules to return to the conformation at which KdME is 200 microM was found to be 60 min. Finally it was shown that the high-affinity binding site for Mg2+ is not formed at low (4 degrees C) temperature.
A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.
There is accumulating evidence that metabolic pathways are organized in vivo as multienzyme clusters or metabolons. To assess interactions between consecutive enzymes of a pathway in vitro, it is usually essential to modify the physical properties of water around the enzymes, e.g. by immobilizing the latter onto a solid support. Such immobilized enzyme preparations can be embedded in agarose gels and used for affinity electrophoresis [Beeckmans, S., Van Driessche, E. & Kanarek, L. (1989) Eur. J. Biochem. 183, 449-454; Beeckmans, S., Van Driessche, E. & Kanarek, L. (1990) J. Cell. Biochem. 43, 297-306]. In this study we use the aforementioned technique to investigate the association between two plant glyoxylic acid cycle enzymes, i.e. isocitrate lyase and malate synthase. A specific histochemical staining technique is described for both enzymes. Affinity electrophoresis using either isocitrate lyase or malate synthase as the immobilized enzyme clearly shows that associations are formed between both enzymes. Moreover, experiments with metabolically unrelated enzymes prove that the observed interaction is specific.
In recent years it has become clear that a cell cannot be visualized as a 'bag' filled with enzymes dissolved in bulk water. The aqueous-phase properties in the interior of a cell are, indeed, essentially different from those of an ordinary aqueous solution. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as 'metabolons'. Very often interactions between enzymes of a pathway can exclusively be evidenced in vitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glycolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate-malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques are also discussed.
A purification scheme is described for the glyoxylate cycle enzyme malate synthase from maize scutella. With our procedure, large amounts of extremely pure enzyme can easily be prepared. Purification involves a heat denaturation step, followed by ammonium sulfate precipitation, and chromatography on DEAE-cellulose and Blue Dextran-Sepharose. Catalase and malate dehydrogenase, which are the most persistent contaminants, are completely removed by this procedure. Maize malate synthase is an octameric protein with a subunit molecular weight of 64 kDa. Purity of the enzyme preparation was demonstrated by SDS-polyacrylamide gel electrophoresis and by isoelectric focusing (pI = 5.0). Pure malate synthase can be stored without appreciable loss of activity at -70 degrees C in 200 mM Hepes buffer containing 6 mM MgCl2 and 2 mM 2-mercaptoethanol, pH 7.6. Maize malate synthase contains no covalently linked carbohydrate residues. The enzyme requires Mg2+ ions for activity. From circular dichroism measurements we estimate that the secondary structure of the enzyme consists of 30% alpha-helical and almost no (5%) beta-pleated sheet segments. A 45-kDa polypeptide, which contaminates malate synthase preparations if the purification starts from seedlings older than 2.5 days, is shown to be a degradation product of malate synthase. Together with full-length chains, these 45-kDa polypeptides are able to take part in octameric oligomer formation.
A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.
In recent years, evidence has been accumulating that metabolic pathways are organized in vivo as multienzyme clusters. Affinity electrophoresis proves to be an attractive in vitro method to further evidence specific associations between purified consecutive enzymes from the glycolytic pathway on the one hand, and from the citric acid cycle on the other hand. Our results support the hypothesis of cluster formation between the glycolytic enzymes aldolase, glyceraldehydephosphate dehydrogenase, and triosephosphate isomerase, and between the cycle enzymes fumarase, malate dehydrogenase, and citrate synthase. A model is presented to explain the possibility of regulation of the citric acid cycle by varying enzyme-enzyme associations between the latter three enzymes, in response to changing local intramitochondrial ATP/ADP ratios.
Evidence is growing that the citric acid cycle, like many other metabolic pathways, might exist in vivo as a more or less tightly organized multi-enzyme cluster. The term 'metabolon' [Robinson, J. B. & Srere, P. A. (1985) J. Biol. Chem. 260, 10800-10805] was recently introduced to describe such a complex of sequential metabolic enzymes. We adopted the technique of affinity electrophoresis for the study of interactions between the cycle enzymes fumarase and malate dehydrogenase. This approach offers several advantages over our previously described affinity chromatographic technique [Beeckmans, S. & Kanarek, L. (1981) Eur. J. Biochem. 117, 527-535], one of which is the fact that the interaction can be directly visualized. The observed association is specific since both metabolically unrelated proteins and the cytoplasmic isoenzyme of malate dehydrogenase do not interact with fumarase. Several metabolites (citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, Pi, AMP, ADP, NAD+, NADH) were found not to affect the association between fumarase and mitochondrial malate dehydrogenase. Both ATP, Mg2+ -ATP and GTP disrupt the association when they are present at 1 mM concentrations. Lower non-physiological ATP concentrations do not, however, disturb the interaction. The presence of 1 mM ADP was found to abolish the disrupting effect of 1 mM ATP. The latter findings are suggestive of an interruption of the citric acid cycle at the level of fumarase under conditions of high energy load (i.e. high ATP/ADP ratios).
Recently we reported the isolation and partial biochemical characterization of the novel polypeptide h3 from human brain and liver. In this report, the physicochemical characterization is further established by the use of several analytical methods. The following results were obtained: the ultraviolet absorption spectrum is not influenced by pH, and the circular dichroism (CD) spectrum reveals that this protein has no alpha-helices, whereas approximately 25% of the polypeptide chain is found to be folded as a beta-pleated sheet structure. Neither the conformation of h3 as assessed by CD nor the titration kinetics of sulfhydryl groups with Ellman's reagent are affected by the presence of the ions K+, Na+, Ca2+, and Mg2+. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a beta-mercaptoethanol gradient and Cleveland sequential SDS-PAGE showed that the frequent formation of h3 polymers and doublets, as observed earlier, is almost exclusively due to disulfide bonding.
alpha 2-Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated agonist p-azido[3H]clonidine. Saturation binding in the dark occurs with high affinity (1.3 +/- 0.3 nM) to a single class of sites (1122 +/- 67 fmol/mg protein). Irradiation of the membrane-bound radioligand results in the labeling of a peptide band with an apparent size of 65 kDa and a characteristic pharmacological profile for an alpha 2-adrenergic receptor. The carbohydrate moieties of the alpha 2-receptor are characterized by lectin affinity chromatography and glycosidase treatment. The Nonidet P-40-solubilized, p-azido[3H]clonidine-labeled receptors are completely retained by Con A- as well as WGA-Sepharose columns. Neuraminidase, alpha-mannosidase and TFMS do not affect the electrophoretic mobility of the receptor on SDS-PAGE whereas endoglycosidase F reduces the apparent size to 45 kDa.
A general analysis of the regulation of the citric acid cycle is hampered by the intimate interplay believed to exist between the various surrounding pathways. Two main regulatory mechanisms are thought to determine the flux through the cycle: (1) regulation of individual cycle enzymes, and (2) reversible complex formation between various enzymes of the cycle and related pathways. The latter mechanism allows a cell to maintain a high flux of substrates with a moderate number of intermediates, and offers a means of metabolite channeling. We were able to demonstrate specific interactions between several vertebrate cycle enzymes in conditions of reduced water concentration, i.e. by using immobilized enzyme systems. From affinity chromatographic experiments, we have shown that the enzymes of the citric acid cycle and the aspartate-malate shuttle are organized as one huge multi-enzyme complex, and a stoichiometric arrangement of fumarase/malate dehydrogenase/citrate synthase/aspartate aminotransferase has been postulated. Affinity electrophoresis was used as a new experimental device by which the enzyme-enzyme interactions could be directly visualized.
Metabisulfite, diethyldithiocarbamate, and thiourea are potent phenoloxidase inhibitors commonly used during the extraction of plant proteins. Their effects on several amino acid derivatives and peptides are reported. Important and pH-dependent changes are induced by metabisulfite in the uv-absorption spectrum of N-acetyltryptophanamide but not of N-acetyltyrosinamide. Similar spectral changes are also induced in tryptophanyl-containing peptides. Neither diethyldithiocarbamate nor thiourea modified the spectral properties of tryptophan or tyrosine. Cysteinyl groups are rapidly modified by diethyldithiocarbamate, especially in the lower pH range, but not by metabisulfite or thiourea. Diethyldithiocarbamate as well as metabisulfite interfere with cysteinyl groups. It is concluded that thiourea is the most appropriate reagent for use as a phenoloxidase inhibitor.
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Modification of pig heart fumarase (L-malate hydro-lyase, EC 4.2.1.2) with tetranitromethane results in loss of enzymatic activity. The inactivation is slowed down in the presence of substrates, indicating that the modification reaction takes place at the level of the substrate binding sites. From these inactivation kinetics, a value Kd = 78 microM is calculated for the mixture of substrates (L-malate + fumarate). This is in fairly good agreement with the Michaelis constant Km = 31 microM. Spectrophotometric data indicate that modification of one tyrosine residue per fumarase subunit is responsible for the inactivation; one or more additional residues, which do not participate in the binding sites, are modified at much lower rates. Amino acid analyses confirm the presence of nitrotyrosine and exclude the possibility of tetranitromethane-mediated polymerization side-reactions. It is concluded from the pH-dependence of the nitration reaction that the inactivation of fumarase is not caused by cysteine modification. Additional studies of nitration of melittin, a tryptophan-containing model peptide, are described. From the absorption spectra of modified melittin, in comparison with the spectra of nitrofumarase, it is concluded that the tryptophan residues of the latter enzyme remain intact during the reaction with tetranitromethane. Finally, evidence is given for an independent action of the four fumarase subunits, i.e., inactivation of one subunit does not influence the catalysis by the other three subunits. Moreover, it is shown that only fumarase tetramers with all four subunits nitrated are unable to bind to a Sepharose-pyromellitic acid affinity column.