Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle.

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 x 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 x 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.     

Aldolase sequesters WASP and affects WASP/Arp2/3‐stimulated actin dynamics

In addition to its roles in sugar metabolism, fructose‐1,6‐bisphosphate aldolase (aldolase) has been implicated in cellular functions independent from these roles, termed “moonlighting functions.” These moonlighting functions likely involve the known aldolase–actin interaction, as many proteins with which aldolase interacts are involved in actin‐dependent processes. Specifically, aldolase interacts both in vitro and in cells with Wiskott–Aldrich Syndrome Protein (WASP), a protein involved in controlling actin dynamics, yet the function of this interaction remains unknown. Here, the effect of aldolase on WASP‐dependent processes in vitro and in cells is investigated. Aldolase inhibits WASP/Arp2/3‐dependent actin polymerization in vitro. In cells, knockdown of aldolase results in a decreased rate of cell motility and cell spreading, two WASP‐dependent processes. Expression of exogenous aldolase rescues these defects. Whether these effects of aldolase on WASP‐dependent processes were due to aldolase catalysis or moonlighting functions is tested using aldolase variants defective in either catalytic or actin‐binding activity. While the actin‐binding deficient aldolase variant is unable to inhibit actin polymerization in vitro and is unable to rescue cell motility defects in cells, the catalytically inactive aldolase is able to perform these functions, providing evidence that aldolase moonlighting plays a role in WASP‐mediated processes. J. Cell. Biochem. 114: 1928–1939, 2013. © 2013 Wiley Periodicals, Inc.     

Aldolase-localization in cultured cells: cell-type and substrate-specific regulation of cytoskeletal associations.

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20-2000 microM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20-60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin-aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.     

Supramolecular forms of actin from amoebae of Dictyostelium discoideum.

Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.     

Calcium control of Saccharomyces cerevisiae actin assembly.

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.     

Inhibition of proteinase K by phosphorylated sugars.

Proteolysis of lactate dehydrogenase, aldolase and the synthetic substrate N-succinylalanylalanylalanyl-p-nitroanilide by proteinase K is inhibited by glucose-6-phosphate and fructose-1,6-biphosphate. Analysis of the kinetic data obtained with the synthetic substrate indicates that the inhibition is a mixed-type and that more than one inhibitor molecule binds to proteinase K. Glucose and fructose are ineffective as inhibitors. In the presence of 0.2-4 mM fructose-1,6-biphosphate, aldolase becomes more susceptible to proteolysis, probably as a result of a conformational change induced by the substrate.     

Characterization of an aldolase-binding site in the Wiskott-Aldrich syndrome protein

The thrombospondin-related anonymous protein (TRAP) is an essential transmembrane molecule in Plasmodium sporozoites. TRAP displays adhesive motifs on the extracellular portion, whereas its cytoplasmic tail connects to actin via aldolase, thus driving parasite motility and host cell invasion. The minimal requirements for the TRAP binding to aldolase were scanned here and found to be shared by different human proteins, including the Wiskott-Aldrich syndrome protein (WASp) family members. In vitro and in vivo binding of WASp members to aldolase was characterized by biochemical, deletion mapping, mutagenesis, and co-immunoprecipitation studies. As in the case of TRAP, the binding of WASp to aldolase is competitively inhibited by the enzyme substrate/products. Furthermore, TRAP and WASp, but not other unrelated aldolase binders, compete for the binding to the enzyme in vitro. Together, our results define a conserved aldolase binding motif in the WASp family members and suggest that aldolase modulates the motility and actin dynamics of mammalian cells. These findings along with the presence of similar aldolase binding motifs in additional human proteins, some of which indeed interact with aldolase in pull-down assays, suggest supplementary, non-glycolytic roles for this enzyme.     

Interaction of aldolase with the troponin–tropomyosin complex of bovine muscle (Short Communication)

Ultracentrifugal studies of mixtures of aldolase and the troponin-tropomyosin complex from bovine muscle showed the existence of a labile interaction between these two myofibrillar constituents in imidazole buffers, pH6.8, I 0.02-0.10 (mol/l), and the suppression of the reaction by fructose 1,6-diphosphate. Analysis of the sedimentation-velocity patterns suggests the binding of more than 2 molecules of troponin-tropomyosin/molecule of aldolase. The results illustrate the necessity of considering additional or alternative sites to F-actin to account for the observed binding of aldolase to the thin filaments of skeletal muscle.     

Complete amino acid sequences of the three collagen-like regions present in subcomponent C1q of the first component of human complement.

Possible interactions between polymerized (F-) actin and insulin-storage granules from rat islets of Langerhans were examined in vitro by comparing the sedimentation of the granules in the presence of various actin concentrations. Actin in the concentration range 0.1--0.5 mg/ml produced a retardation in granule-sedimentation rates consistent with binding of the granules to the actin filaments. The interaction was increased by addition of ATP (2mM), but was decreased by CaCl2 (0.1 mM). Binding of granules to actin was unaffected by cyclic AMP or by preincubation of the granules with phospholipase C. Specificity of the interaction was confirmed by the use of depolymerized (G-) actin and of myosin to provide a solution of comparable viscosity; neither of these caused any alteration of granule sedimentation. Possible implications of this interaction of insulin-storage granules with actin for the mechanism of insulin secretion are briefly discussed.     

Properties of two isoforms of human blood platelet alpha-actinin

The structural and functional properties of the aa (2 X 97 kDa) and cc (2 X 94 kDa) isoforms of platelet alpha-actinin have been compared. Structural differences between aa and cc are revealed by their peptide maps, obtained from limited proteolysis, and by their immunological cross-reactivity. Both isoforms stimulate the Mg ATPase activity of actomyosin, bind to F-actin (high-speed sedimentation) and cross-link or gel actin filaments (low-speed sedimentation and viscometry), in a calcium-dependent manner. The study of the interaction with F-actin indicates that the binding of 1 molecule of aa or cc alpha-actinin/9-11 actin monomers is sufficient to produce maximal gelation in the presence of EGTA. CaCl2 at 0.1 mM strongly inhibits the binding of aa to F-actin and weakly that of cc, while it inhibits similarly the cross-linking of either aa or cc. The cross-linking efficiency of cc is 9, 7, 1.7 and 1.3 times higher than that of aa at 4, 20, 30 and 37 degrees C, respectively. The bb form (2 X 96 kDa), which is a proteolytic product of aa [Y. Gache et al. (1984) Biochem. Biophys. Res. Commun. 124, 877-881], behaves roughly as aa, but the calcium sensitivity of its binding to F-actin is intermediate between that of aa and cc. These results suggest that the effect of Ca2+ concentration on the binding of platelet alpha-actinin to F-actin may be partly dissociated from the effect on the cross-linking.     

Intracellular localization of fructose 1,6-bisphosphate aldolase.

Submission of a rat liver homogenate made in 250 mM sucrose-1 mM EDTA to centrifugation between 9,500 times g for 10 min and 105,000 times g for 60 min results in the sedimentation of 60 to 70% of the total cellular fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Under these conditions only about one-quarter of the total triose phosphate dehydrogenase and phosphoglycerate kinase appears in the microsomal fraction. Ultrastructural immunologic localization techniques have demonstrated that the aldolase is associated with the endoplasmic reticulum, in situ. The binding of this enzyme to the membrane is sensitive to changes in pH with an optimum at 6.0, and to increasing concentrations of NaCl and fructose 1,6-bisphosphate, being about 100-fold more sensitive to the ester than to the inorganic salt.     

Ca2+-calmodulin-dependent polymerization of actin by myelin basic protein

The interaction between myelin basic protein (MBP) and G-actin was studied under nonpolymerizing conditions, i.e.,2mM HEPES, pH 7.5, 0.1 mM CaCl2 and 0.2 mM ATP. Fluorescence studies using pyrenyl-actin and the measurements of ATP hydrolysis rate show that MBP induces changes in the structure of the actin monomer similar to those occurring during polymerization by salt. Electron microscope observations of the MBP-G-actin complex reveal the presence of filamentous structures which appear as separate filaments or as bundles of filaments in lateral association. These filaments are polar as visualized by attachment of heavy meromyosin. The biochemical data together with electron microscope observations suggest that the binding of MBP to G-actin under non-polymerizing conditions induces an interaction between actin monomers leading to the formation of filamentous structures which may be similar to F-actin filaments. The effects of MBP on G-actin can be reversed by calmodulin in the presence of Ca2+.     

Influence of ionic strength,actin state,and caldesmon construct size on the number of actin monomers in a caldesmon binding site

There is no consensus on the mechanism of inhibition of actin-myosin ATPase activity by caldesmon. Various models are based on different assumptions for the number of actin monomers that constitute a caldesmon binding site. Differences in binding behavior may be due to variations in the assay, the range of caldesmon concentrations, the type of caldesmon, and the method of data analysis used. We have evaluated these factors by measuring binding in the presence and absence of tropomyosin with both intact caldesmon and a recombinant 35 kDa actin binding fragment and with actin initially in the polymerized state or monomeric state. In all cases caldesmon binding could be simulated with a model having one class of binding sites. However, the number of actin monomers constituting a site was variable. Binding to F-actin at 165 mM ionic strength was best described with 7 actin monomers per site. When caldesmon bound to actin during the polymerization of G-actin, the size of the binding site was 3. Binding of the expressed truncated fragment, Cad35, could be described with 3 monomers per site. A simple interpretation of the data is that caldesmon binds tightly to 2-3 actin monomers. Additional parts of caldesmon bind less tightly to actin, causing caldesmon to cover approximately 7 actin monomers. The appendix contains an analysis of several binding curves with multiple binding site models. There is no compelling evidence for two classes of binding sites.     

Properties of the interaction between phosphofructokinase and actin

The interaction of rabbit skeletal muscle phosphofructokinase (PFK) with actin is characterized in terms of the binding of PFK to actin in the presence and absence of tropomyosin and troponin, the effect of PFK on actin polymerization, and the involvement of adenylates in the binding of PFK to actin. The thin filament proteins, tropomyosin and troponin, are associated with skeletal muscle actin and reduce the binding of PFK to actin, thus influencing the probable distribution of PFK in skeletal muscle. The binding of PFK to actin is inhibited by ATP and ADP but not by fructose 6-phosphate or fructose 2,6-bisphosphate. This specific inhibition, plus evidence from fluorescence quenching and photoaffinity labeling, suggests that actin binds at the adenosine activation sites of PFK. Light scattering measurements used to monitor actin polymerization indicate that PFK dramatically increases the level of light scattering produced by the polymerization of actin, indicative of a superaggregate of PFK and actin. PFK inhibits the polymerization of actin when polymerization is induced by low concentrations of added salts. Although PFK binds to actin with high affinity, it seems to have little effect on the high shear viscosity of actin filaments.     

Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose-induced acrosin inhibition

Acrosin (acrosomal proteinase; EC 3.4.21.10) is a sperm-specific serine proteinase implicated in sperm penetration of the mammalian oocyte. Previously, we had shown that human acrosin, unlike human trypsin (EC 3.4.21.4), was inhibited by beta-D-fructose and related carbohydrates. The present study was undertaken to more fully elucidate the mechanism of action of fructose as an acrosin inhibitor, and to further differentiate the kinetic properties of acrosin from those of trypsin. Fructose produced a complex pattern of inhibition. At relatively low concentrations (10-60 mM), fructose acted as a competitive inhibitor with an apparent inhibition constant of 13 mM. In contrast, at high concentrations (80-320 mM), fructose behaved as a noncompetitive inhibitor, with an apparent inhibition constant of 205 mM. A Hill plot of enzyme activity as a function of fructose concentration suggested only a single binding site for fructose (slope = -0.90). The pattern of inhibition is not consistent with an enzyme containing only a single catalytic site, based either upon steady-state or rapid equilibrium assumptions; however, good agreement between observed and simulated data were obtained based upon the assumption of two catalytic sites with equal or similar binding and catalytic constants. The data suggested that fructose interacts with a single binding site (Ki = 8 mM) which alters both catalytic sites to produce an enzyme species having a higher apparent Michaelis constant and lower kcat as compared to the uninhibited enzyme. Fructose had no effect upon the rate of acrosin inactivation by either diisopropylfluorophosphate or tosyl-lysine-chloromethylketone, suggesting that neither substrate binding nor acylation were altered by this agent. The above data indicate substantial differences between the catalytic properties of human acrosin and those of trypsin.     

Control of phosphofructokinase from rat skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate.

Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate.     

Interaction of the aldolase and the membrane of human erythrocytes.

Up to 80% of cellular aldolase (EC 4.1.2.13) was retained in the membrane fraction isolated following hemolysis of human erythrocytes under appropriate conditions. Binding was reversed by increasing the pH and ionic strength. Millimolar levels of the substrate, fructose 1,6-bisphosphate, selectively eluted aldolase from the membrane, while related metabolites did not. Using the membrane as a high affinity adsorbant, electrophoretically pure aldolase of high specific activity was prepared in high yield. The reassociation of pure aldolase and membranes was characterized. The sole site of human erythrocyte aldolase binding was shown to be the cytoplasmic surface domain of band 3, the predominant membrane-spanning polypeptide. One aldolase molecule was bound per band 3 polypeptide. Upon binding to either whole membranes, solubilized band 3, or proteolytic fragments from the cytoplasmic surface pole of band 3, aldolase underwent a profound loss of catalytic activity, reversed by raising the substrate concentration.     

Evidence for an interaction between fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase.

Three distinct lines of evidence suggest interaction and possible complex formation between fructose 1,6-biphosphate aldolase (EC 4.1.2.13) and fructose 1,6-biphosphatase (EC 3.1.3.11) from rabbit liver. (1) Fructose 1,6-biphosphatase, which does not contain tryptophan, causes changes in the fluorescence emission spectrum of tryptophan in rabbit liver aldolase. (2) Aldolase reduces the affinity of binding of Zn²⁺ to the two high-affinity sites of fructose 1,6-biphosphatase. (3) Gel penetration coefficients are decreased for both enzymes when they are tested together, as compared to the coefficients observed when each is tested separately. These interactions were not observed when either liver enzyme was replaced by the corresponding enzyme purified from rabbit muscle; this specificity for enzymes purified from the same tissue excludes effects attributable to the catalytic activities of the enzyme. Maximum interaction was observed in the pH range between 8.0 and 8.5 and appeared to require the presence of two fructose 1,6-biphosphatase tetramers per tetramer of aldolase. The change in fluorescence emission spectrum was also observed, to a smaller extent, when muscle fructose 1,6-biphosphatase was added to a solution of muscle aldolase.     

Preparation of monomeric cytochrome C oxidase: its kinetics differ from those of the dimeric enzyme

Bovine cytochrome c oxidase in 0.1% dodecylmaltoside, 50 mM KCl and 10 mM Tris-HCl, pH 7.4 is monodisperse with an apparent Mr 360,000 (dimer) as estimated by filtration on Ultrogel AcA 34. In the absence of added KCl the apparent Mr is 160,000 (monomer). The dimeric enzyme has a high and a low affinity site for cytochrome c; the monomeric, only the high affinity site. The results are consistent with the existence of one active site per monomer, having high affinity for cytochrome c. Since in a dimer the two sites are in close proximity, the binding of the first molecule of cytochrome c to the first site hinders the binding of the second molecule to the second site. The kinetic data fit with a model of homotropic negative cooperativity. The effect of salts on the cytochrome c oxidase kinetics is also present in isolated bovine heart mitochondria.     

Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex--purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.