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PubMed1987

Fluorescence investigation on conformational state of rabbit muscle aldolase interacting with phosphatidylinositol liposomes.

Evidence of conformational changes in rabbit muscle aldolase upon binding to phosphatidylinositol liposomes and the effect of the interaction on the thermal conformational transition are reported. Interaction with phosphatidylinositol liposomes significantly decreases the aldolase tryptophanyl fluorescence and shifts the maximum wavelength to higher values. The dynamic quenching constant for the aldolase fluorescence quenching by acrylamide in the presence of liposomes is much higher than that for unmodified enzyme; this signifies an increase in accessibility of some tryptophanyl residues to small polar molecules. Indirect interaction between single phospholipid molecules, small micelles or any soluble impurities able to penetrate into the protein molecule interior does not seem to be involved in the conformational rearrangement. Native and liposome-interaction-induced conformational states reveal different temperature dependences of the tryptophan residues exposure. The implications of the modification of the conformational state of the enzyme for its function in vivo are discussed.

Animals↗
PubMed1976

Aldolase B in the liver of senescent rats.

The process of enzymatic aging was studied in livers of adult and senescent rats for aldolase B. No "cross-reacting material" was found in livers of 27 to 30-month-old rats, estimated by the ratio aldolase activity-antigen amount. The activity towards the two substrates of aldolase, fructose 1,6-diphosphate and fructose 1-phosphate did not vary in senescent animals. Moreover, other physico-chemical properties of the enzyme such as thermal inactivation, immunological reactivity and Michaelis constant remain unchanged. These results provide arguments against the occurrence of errors in protein synthesis as a cause of aging.

Aging↗
PubMed1983

Studies on the fate of aldolase molecules in the aging rat lens.

It was found that aldolase activity declined considerably in the lens of adult animals with increasing age. Immunoassay showed that defective aldolase C molecules were accumulated. In addition, antibody prepared against denatured enzyme preferentially removes inactive molecules from lens homogenates without affecting active molecules. It is concluded that defective aldolase molecules encountered in aging lenses are at least partially denatured and are inactive.

Aging↗
PubMed1985

Isolation and characterization of pig muscle aldolase. A comparative study.

Aldolase with a specific activity of 10.8 units/mg protein was isolated from pig muscle. Its molecular weight was found to be 150,000. The optimum pH for the catalytic activity was 7.25 and the apparent temperature optimum was 313 K. The Km value was 2.9 X 10(-5) M with FDP as substrate, and 2.8 X 10(-3) M with F1P as substrate. The thermal stability of this pig muscle enzyme was higher than that of the rabbit muscle enzyme. The thermal inactivation of the pig aldolase did not show simple first-order kinetics. The higher conformational stability of the pig aldolase than that of the rabbit enzyme was demonstrated by its higher resistance to the denaturing effect of urea.

Animals↗
PubMed2005

The effect of calcium ions on subcellular localization of aldolase-FBPase complex in skeletal muscle.

In skeletal muscles, FBPase-aldolase complex is located on alpha-actinin of the Z-line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase-aldolase and FBPase-alpha-actinin complex, causes fast dissociation of FBPase from the Z-line and slow accumulation of aldolase within the I-band and M-line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis.

Actinin↗
PubMed2004

Molecular cloning, expression, purification, and characterization of fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis--a novel Class II A tetramer.

The Class II fructose 1,6-bisphosphate aldolase (fda, Rv0363c) from the pathogen Mycobacterium tuberculosis H37RV was subcloned in the Escherichia coli vector pT7-7 and purified to near homogeneity. The specific activity (35 U/mg) is approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen. Attempts to express the enzyme with an N-terminal fusion tag yielded inactive, mostly insoluble protein. The native recombinant enzyme is zinc-dependent and has a catalytic efficiency for fructose 1,6-bisphosphate cleavage higher than most Class II aldolases characterized to date. The aldolase has a Km of 20 microM, a kcat of 21 s(-1), and a pH optimum of 7.8. The molecular mass of the enzyme subunits as determined by mass spectrometry is in agreement with the mass calculated on the basis of its gene sequence minus the terminal methionine, 36,413 Da. The enzyme is a homotetramer and retains only two zinc ions per tetramer when transferred to a metal-free buffer, as determined by ICP-MS and by a colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) as a chelator. The E. coli expression system reported in this study will facilitate the further characterization of this enzyme and the screening for potential inhibitors.

Bacterial Proteins↗
PubMed1984

Different expression of rat aldolase A mRNA in the skeletal muscle and ascites hepatoma cells.

Rat aldolase A mRNAs from the skeletal muscle and ascites hepatoma cells are compared by RNA blot hybridization. The hepatoma AH60C and AH130 mRNA are about 1600 bases long, some 50 bases longer than the skeletal muscle transcript. Aldolase A enzymes encoded by these mRNAs, however, are indistinguishable in electrophoresis on cellulose polyacetate strips and two-dimensional polyacrylamide gel. In addition, comparison of the thermal stability of hybrids between the mRNAs from the skeletal muscle and hepatoma cells and a cDNA probe (pRAAM83) made from the aldolase A mRNA of the skeletal muscle indicate that the mRNAs have extremely high sequence homology, at least, within the most part of coding region in addition to the 3' noncoding region. These findings indicate that these mRNA may be transcribed by the use of different promoter sites and the common structural gene.

Animals↗
PubMed1987

Aldolase decreases the dissociation-induced inactivation of muscle phosphofructokinase.

The effect of aldolase on the concentration-dependent kinetic behaviour of phosphofructokinase was investigated by means of covalently attached fluorescent probe and by using a kinetic approach. The dimeric form of kinase in equilibrium with the active tetramer interacts with the native aldolase with an apparent dissociation constant of 2.5 microM. Within this heterologous enzyme complex the phosphofructokinase is catalytically active probably because the aldolase binding to nascent kinase dimers might protect them against inactivation.

Allosteric Regulation↗
PubMed2003

Order, disorder, and perturbations in actin-aldolase rafts.

Actin-aldolase rafts provide insights into the use of rafts as models for three-dimensional actin bundles. Although aldolase has three twofold axes, filaments in actin-aldolase rafts were not strictly related by a twofold axis. Interfilament angles were on average +15 degrees off the expected 180 degrees, and most rafts appeared handed; that is, rows of cross-bridges were tilted in a clockwise direction off the perpendicular. We can account for both the deviation of the angle from 180 degrees and the handedness of the rafts by a steric constraint due to the lipid layer. We further found that the axial spacings of cross-bridges varied significantly from raft to raft. We suggest that this difference arises from variations in the twist of the filaments that nucleate raft formation; that is, filaments added to a raft adopt the symmetry of those in the raft. We conclude that the organization of filaments in rafts can be modulated by outside factors such as the lipid layer and that the variable twist of filaments in the nucleating core of the raft are imposed on all the filaments in the raft. These results provide a measure of the potential for polymorphism in actin assemblies.

Actins↗
PubMed2003

Muscle FBPase in a complex with muscle aldolase is insensitive to AMP inhibition.

Real-time interaction analysis, using the BIAcore biosensor, of rabbit muscle FBPase-aldolase complex revealed apparent binding constant [K(Aapp)] values of about 4.4x10(8) M(-1). The stability of the complex was down-regulated by the glycolytic intermediates dihydroxyacetone phosphate and fructose 6-phosphate, and by the regulator of glycolysis and glyconeogenesis--fructose 2,6-bisphosphate. FBPase in a complex with aldolase was entirely insensitive to inhibition by physiological concentrations of AMP (I(0.5) was 1.35 mM) and the cooperativity of the inhibition was not observed. The existence of an FBPase-aldolase complex that is insensitive to AMP inhibition explains the possibility of glycogen synthesis from carbohydrate precursors in vertebrates' myocytes.

Adenosine Monophosphate↗
PubMed1999

Chloroplast class I and class II aldolases are bifunctional for fructose-1,6-biphosphate and sedoheptulose-1,7-biphosphate cleavage in the Calvin cycle.

Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6- and sedoheptulose-1,7-P2 in the Calvin cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6- and sedoheptulose-1,7-P2. Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type.

Cell Compartmentation↗
PubMed1997

Structure of aldolase A (muscle-type) cDNA and its regulated expression in oocytes, embryos and adult tissues of Xenopus laevis.

We obtained cDNA (XALDA; 1466 bp) for Xenopus laevis aldolase A gene (muscle-type), whose amino acid sequence had 88% similarity to those of mammalian aldolase A genes. XALDA mRNA occurred abundantly in skeletal muscle and at low levels also in other adult tissues, and such mRNA distribution was reflected in zymograms. In oocytes XALDA mRNA occurred at a relatively high level from stage I, and the mRNA level peaked at stage II, then decreased in later stages. XALDA mRNA in the full-grown oocyte was inherited as maternal mRNA throughout maturation and fertilization until midblastula stage, but its level became very low during gastrula and early neurula stages, and then increased greatly in later stages. While maternal XALDA mRNA was distributed uniformly in early embryos, mRNA zygotically expressed after late neurula stage occurred mainly in somites. In blastula animal caps XALDA mRNA occurred at a low level, but the expression was greatly enhanced by activin treatment. Thus, in Xenopus laevis aldolase A gene is actively transcribed in earlier phase of oogenesis, inherited as maternal mRNA in early embryos in a cell-type nonspecific way, then in later phases of embryogenesis, it is strongly expressed in somites with its concomitant ubiquitous expression at low levels in almost all the other cell types.

Activins↗
PubMed1999

Functional studies by site-directed mutagenesis on the role of Sp1 in the expression of the pyruvate kinase M and aldolase A genes.

During the cell cycle of mitogen stimulated rat thymocytes, an 8-10-fold induction of glycolytic enzymes and a corresponding increase in the mRNA levels has been observed. This prompted us to study the transcriptional regulation of the rat aldolase A and pyruvate kinase M genes. cis-Regulatory elements of both promoters were evaluated by site-directed mutagenesis of promoter/luciferase constructs and transient transfections of rat hepatoma FTO2B cells. Furthermore, the binding proteins were identified by mobility shift assays in the presence of specific antibodies. In the aldolase AH1 promoter, five binding sites for Sp1 and Sp3 and a TPA responsive element were identified as essential for transcriptional regulation. Most of the promoter activity can be attributed to these regulatory elements. In the pyruvate kinase M promoter three out of five binding sites of Sp1 and Sp3 (B box and GC boxes 1 and 3) turned out to be functional in the transfection assays whereas the disruption of GC box 2 had no effect, and the disruption of the GC box 4 had only a minor effect on the promoter activity. Both promoters are stimulated by Sp1 as well as Sp3, as judged by cotransfection experiments of Drosophila SL2 cells. Therefore, the Sp1- and Sp3-directed transcription provides a means for common regulatory mechanism of the aldolase A and the pyruvate kinase M genes.

Animals↗
PubMed2003

Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites.

Host cell invasion by apicomplexan parasites requires coordinated interactions between cell surface adhesins and the parasite cytoskeleton. We have identified a complex of parasite proteins, including the actin binding protein aldolase, which specifically interacts with the C-terminal domains of several parasite adhesins belonging to the thrombospondin-related anonymous protein (TRAP) family. Binding of aldolase to the adhesin was disrupted by mutation of a critical tryptophan in the C domain, a residue that was previously shown to be essential for parasite motility. Our findings reveal a potential role for aldolase in connecting TRAP family adhesins with the cytoskeleton, and provide a model linking adhesion with motility in apicomplexan parasites.

Actin Cytoskeleton↗
PubMed1994

Lysine-146 of rabbit muscle aldolase is essential for cleavage and condensation of the C3-C4 bond of fructose 1,6-bis(phosphate).

Lysine-146 of rabbit muscle aldolase (D-fructose-1,6-biphosphate aldolase, EC 4.1.2.13) is absolutely conserved in class I (Schiff base) aldolases and has been implicated previously in catalysis by protein modification. Site-directed mutagenesis was used to change lysine-146 to alanine, glutamine, leucine, or histidine, creating the mutant enzymes K146A, K146Q, K146L, and K146H, respectively. These mutant proteins were expressed at high levels in bacteria and were purified by substrate affinity elution from CM-Sepharose, the same method that is used for the wild-type enzyme. The mutants K146A, K146Q, and K146L had substrate cleavage rates below standard detection levels. Modified cleavage assays indicated that these enzymes were (0.5-2) x 10(6)-fold decreased in the rate of catalysis of fructose 1,6-bis(phosphate) (Fru-1,6-Pa)cleavage. The K146H enzyme, however, was approximately 2000-fold slower than wild type in the rates of both cleavage and condensation of Fru-1,6-P2. In assays for the presence of enzymatic intermediates, all of the mutant enzymes were able to catalyze formation of the carbanion intermediate with dihydroxyacetone phosphate, whereas this intermediate was below the level of detection with Fru-1,6-P2. Single-turnover experiments with these enzymes in excess over radiolabeled Fru-1,6-P2 were used to measure the rates of Schiff base and product formation. The rate of Schiff base formation was decreased in each of the mutant enzymes, yet the magnitude of this decrease was less than the reduction in the respective kcat. These mutations had a much larger effect, however, on the rate of C3-C4 bond breaking, showing that Lys-146 is crucial at this step of the catalytic cycle.

Animals↗
PubMed1983

Role of mono- and divalent metal cations in the catalysis by yeast aldolase.

The rate of deuterium exchange between [1-(S)-2H]dihydroxyacetone 3-phosphate and the solvent catalyzed by native and metal-substituted yeast aldolases has been measured. In the presence of 0.1 M potassium acetate at 15 degrees C, pH 7.3, the deuterium exchange reaction catalyzed by native yeast aldolase has a kcat of 95 s-1. In contrast to the 7-fold activity enhancement by 0.1 M potassium ion (relative to 0.1 M sodium ion) of the cleavage of D-fructose 1,6-bisphosphate catalyzed by native yeast aldolase, a negligible (1.1-fold) activation by 0.1 M potassium ion is observed in the rate of dedeuteration of [1(S)-2H]dihydroxyacetone 3-phosphate. The order of reactivity of the yeast metalloaldolases in the deuterium exchange roughly parallels that seen in the fructose bisphosphate cleavage reaction. These findings suggest that the carbonyl groups of enzyme-bound D-fructose 1,6-bisphosphate and dihydroxyacetone phosphate are both polarized by the active site divalent metal cation. A mechanistic formulation consistent with the results of this and the previous paper is presented.

Deuterium↗
PubMed1981

Concentration of activated intermediates of the fructose-1,6-bisphosphate aldolase and triosephosphate isomerase reactions.

As discovered by Grazi & Trombetta [Grazi, E., & Trombetta, G. (1978) Biochem. J. 175, 361], fructose-1,6-bisphosphate aldolase of rabbit muscle causes the slow formation of inorganic phosphate (Pi) and methylglyoxal when incubated with dihydroxyacetone phosphate (DHAP). In addition, these authors found an acid-labile intermediate in equilibrium with the aldolase-dihydroxyacetone phosphate complexes representing approximately 60% of the enzyme-bound DHAP species. Experiments are reported here which argue that this acid-labile species is the enzyme-bound enamine phosphate or its equivalent that decomposes by beta elimination in acid. A similar mechanism involving an enediol phosphate is proposed to explain a phosphatase action of triosephosphate isomerase that produces methylglyoxal and Pi at the rate of approximately 0.1 s(-1) at pH 5.5. When DHAP with excess isomerase is quenched in strong acid, the formation of Pi indicates that approximately 5% of bound reactant is in the form of enediol phosphate. The remainder of the substrate is about equally distributed between bound forms of DHAP and D-glyceraldehyde 3-phosphate. This equilibrium differs by 300-fold from the appropriate equilibrium in solution. Yeast aldolase, contrary to expectation, does not catalyze formation of inorganic phosphate and methylglyoxal when incubated with DHAP and gives no evidence fro an enediol phosphate intermediate when quenched in acid.

Animals↗
PubMed1976

Fructose 1,6-bisphosphate: isomeric composition, kinetics, and substrate specificity for the aldolases.

13C NMR shows fructose 6-phosphate and fructose 1,6-bisphosphate to contain respectively 4.1 and 2.0% keto isomer at room temperature. The lower value for fructose 1,6-bisphosphate can be attributed to the electron-withdrawing effect of the C-1 phosphate. Measurements of the ring-opening rates of the alpha and beta anomers of fructose, 1,6-bisphosphate by an NMR line-broadening technique show them to be about 8 and 35 S-1, respectively, at pH 7.2, and 25degreesC. The value for the predominant beta anomer is threefold greater than the turnover rate of muscle aldolase so that, if the kinetic properties of the keto form were favorable, the reaction could proceed entirely through the keto form in solution. The kinetic properties of a fructose 1,6-bisphosphate(keto) analogue, 5-deoxyfructose, 1,6-bisphosphate, in the muscle aldolase reaction are more favorable (Vmax = 2.6, Km = 0.11 X 10(-6) M) than those of fructose 1,6-bisphosphate total (Vmax = 1, Km = 2.3 X 10(-6)M), giving a value of Vmax/Km that is 56 times greater for the 5-deoxy analogue. At the 2.0% concentration of the keto form this is sufficient to account for the steady-state rate and requires that the beta form, present at 40 times greater concentration, contributes little to the cleavage rate. With yeast aldolase the cleavage rate can be explained by the rapid spontaneous ring opening and reaction of the keto form with the enzyme. In view of the high rate of ring opening and the excellent properties of the keto form, previous rapid kinetic studies favoring action of cyclic forms may require reevaluation.

Fructose-Bisphosphate Aldolase↗