Subcellular localization of aldolase B

The localization of the aldolase B isozyme was determined immunohistochemically in rat kidney and liver using a polyclonal antibody. Aldolase B was preferentially localized in a nuclear region of hepatocytes from the periportal region and was absent in those from the perivenous region. Aldolase B was also preferentially localized in the proximal tubules and was absent in other structures of the renal cortex as well as in the renal medulla. Using reflection confocal microscopy, the enzyme was preferentially localized in a nuclear position in liver and renal cells, which was similar to the cellular and intracellular location found for the gluconeogenic enzyme fructose-1,6-bisphosphatase (Sáez et al. [1996] J. Cell. Biochem. 63:453-462). Subcellular fractionation studies followed by enzyme activity assays revealed that aldolase activity was associated with subcellular particulate structures. Overall, the data suggest that different aldolase isoenzymes are needed in the glycolytic and gluconeogenic pathways.     

The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones

The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81% amino acid homology with aldolase A and 70% homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons separated by eight introns, all in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.     

Thermodynamic analysis shows conformational coupling and dynamics confer substrate specificity in fructose-1,6-bisphosphate aldolase

Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifying and characterizing enzyme dynamics are critical for understanding catalytic mechanisms. Herein, coupling analysis, which uses thermodynamic analysis to assess cooperativity and coupling between distal regions on an enzyme, is used to interrogate substrate specificity among fructose-1,6-(bis)phosphate aldolase (aldolase) isozymes. Aldolase exists as three isozymes, A, B, and C, distinguished by their unique substrate preferences despite the fact that the structures of the active sites of the three isozymes are nearly identical. While conformational flexibility has been observed in aldolase A, its function in the catalytic reaction of aldolase has not been demonstrated. To explore the role of conformational dynamics in substrate specificity, those residues associated with isozyme specificity (ISRs) were swapped and the resulting chimeras were subjected to steady-state kinetics. Thermodynamic analyses suggest cooperativity between a terminal surface patch (TSP) and a distal surface patch (DSP) of ISRs that are separated by >8.9 A. Notably, the coupling energy (DeltaGI) is anticorrelated with respect to the two substrates, fructose 1,6-bisphosphate and fructose 1-phosphate. The difference in coupling energy with respect to these two substrates accounts for approximately 70% of the energy difference for the ratio of kcat/Km for the two substrates between aldolase A and aldolase B. These nonadditive mutational effects between the TSP and DSP provide functional evidence that coupling interactions arising from conformational flexibility during catalysis are a major determinant of substrate specificity.     

Interaction of rabbit muscle aldolase at high ionic strengths with vanadate and other oxoanions.

Reductive, nonreductive, and photolytic interactions of vanadate with fructose-1,6-bisphosphate aldolase were examined and used to explore the interactions of oxoanions with aldolase. Aldolase is known to interact strongly with oxoanions at low ionic strength and weakly at higher ionic strength. Oxoanions inhibit aldolase competitively with respect to fructose 1,6-bisphosphate although the location of the oxoanion binding site on aldolase remains elusive. In this work, the interaction of aldolase with a series of oxoanions was compared at ionic strength approaching physiologic levels. The size and shape of the anion were important for the effective binding to aldolase, and no significant increase in affinity for aldolase was observed by the addition of alkyl groups to the oxoanions. Vanadate competitively inhibits aldolase in a manner analogous to the other oxoanions. Since vanadate solutions contain a mixture of vanadate oxoanions, the nature of the inhibition was determined using a combination of enzyme kinetics and 51V NMR spectroscopy. Aldolase contains a significant number of thiol functionalities, and as expected, vanadate undergoes redox chemistry with them, generating an irreversibly inhibited aldolase. This oxidative chemistry was attributed to the vanadate tetramer, whereas vanadate dimer was a reversible inhibitor. Vanadate monomer does not significantly interact with aldolase reversibly or irreversibly. Vanadyl cation has the lowest inhibition constant under these high ionic strength conditions. Using Yonetani-Theorell analysis, it appears that phosphate, pyrophosphate, and sulfate bind to the same site on aldolase, whereas vanadate, arsenate, and molybdate bind to another site. UV light-induced photocleavage of aldolase by vanadate was examined, and the loss of aldolase activity was correlated with cleavage of the aldolase subunit. Further studies using vanadium as a probe should reveal details on the location of the vanadate and vanadyl cation binding sites. This study suggests several sites on aldolase will accommodate oxoanions, and one of these sites also accommodates vanadyl cation.     

Isozyme differentiation of aldolase and pyruvate kinase in fetal,regenerating, preneoplastic,and malignant rat hepatocytes during culture

Summary Aldolase and pyruvate kinase isozymes were investigated in cultured hepatocytes from fetal, regenerating, and 2-acetyl-aminofluorene-fed rat liver as well as in some epithelial liver cell lines. Our results show that: (a) cell proliferation and prolonged expression of specific isozymes were found only in cultured hepatocytes from 17-day old fetuses; (b) the fetal type of pyruvate kinase expressed in regenerating and carcinogen-treated liver was temporarily lost only in cultured hepatocytes from regenerating liver; (c) the adult type of aldolase and pyruvate kinase was absent in one epithelial cell line derived from a carcinogen-treated liver and in the hepatoma tissue cell (HTC) line but was found in the Faza clone of the Reuber H₃₅ cell line during the 50 first passages in vitro; and (d) the isozyme pattern of pyruvate kinase was always more strongly shifted than that of aldolase. The observations suggest that: (a) hepatocytes from carcinogen-treated liver exhibit the same lack of ability to proliferate in primary culture as normal adult hepatocytes; (b) adult hepatocytes can produce fetal isozymes without prior cell division; (c) pyruvate kinase is a stronger marker of dedifferentiation (retrodifferentiation) than aldolase; and (d) regulatory processes of isozyme expression are different during ontogenesis, regeneration, and hepatocarcinogenesis. This work was supported by the “Institut National de la Santé et de la Recherche Médicale” and the “Fondation pour la Recherche Medicale Fran?aise”     

Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection

Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The k(cat) values of the antibody-catalyzed retro-aldol reactions were correlated with the K(d) values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower K(d) value displayed a higher k(cat) value in the retro-aldol reaction, and a linear relationship was observed in the plots of logk(cat) versus logK(d). These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities.     

Hybridization between fructose diphosphate aldolase subunits derived from diverse biological systems: anomolous hybridization behavior of some aldolase subunit types

In the present studies we investigated the abilities of fructose diphosphate aldolase subunits derived from diverse biological sources to form stable heterotetramers with each other in vitro. Aldolase C subunits isolated from chicken brain readily "hybridized" with aldolase subunits derived from lobster muscle and wheat germ following reversible acid dissociation of mixtures of these enzymes; however, appreciable amounts of stable heterotetramers containing chicken C subunits and aldolase subunits isolated from two other invertebrates (Ascaris and squid) were not produced under the same conditions. In contrast to the situation with chicken C subunits, aldolase B subunits isolated from rat liver did not "hybridize" appreciably with lobster muscle or wheat germ aldolase subunits. The present observations are not consistent with the hypothesis that the abilities of different aldolase subunit types to form heterotetramers in vitro is governed solely by the evolutionary relationships which exist between the organisms from which the enzymes are derived.     

Enzyme activities of human erythrocyte ghosts: effects of various treatments

Summary Ghosts of human erythrocytes prepared by hypotonic hemolysis were assayed for aldolase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase, and glutathione peroxidase and reductase. Cryptic activity of the enzymes was demonstrated by an increase in activity on dilution with water, which caused fragmentation of the ghosts. Aldolase and glyceraldehyde phosphate dehydrogenase were classed as firmly bound; phosphoglycerate kinase was intermediate; the others were loosely bound. Triton X-100 increased the activities of aldolase, glyceraldehyde phosphate dehydrogenase, and phosphoglycerate kinase. The pH of the medium had little effect upon the firmly bound enzymes but it markedly affected the retention of hemoglobin and the activities of the loosely bound enzymes. The presence of Mg or Ca ions enhanced the retention of hemoglobin and the activity of lactate dehydrogenase and pyruvate kinase, with little effect on aldolase and glyceraldehyde phosphate dehydrogenase. Ghosts diluted in water disintegrated into fragments and tubules or vesicles; Mg or Ca at 1mm afforded protection against this. When ghosts were treated with Triton X-100 and adenosine triphosphate, they contracted to about one-seventh of their volume. The shrunken ghosts had lost a considerable proportion of their cholesterol and protein to the medium.     

Differential distribution of aldolase A and C in the human central nervous system

We have analyzed the distribution of aldolase A and C mRNAs and proteins in various areas of the human brain using Northern blot analyses and immunohistochemistry. Aldolase A mRNA expression was higher than aldolase C mRNA expression in all areas of the brain examined. Aldolase C mRNA expression was highest in the cerebellum. Aldolase C protein was present in well-delimited regions of the CNS, and was distributed in stripes in the Purkinje cell layer of the cerebellum, in the inferior olives and in the sensory neurons of the posterior horn of the spinal cord. The novel finding of aldolase C in well-delimited cell compartments of the human cerebellum and in several other areas of the CNS lends weight to the hypothesis that this protein exerts other functions (e.g. sensory transmission) besides those characteristic of a glycolytic enzyme.     

A hydrophobic pocket in the active site of glycolytic aldolase mediates interactions with Wiskott-Aldrich syndrome protein

Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, four-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds around the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C terminus with its active site and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure, a novel naphthol phosphate-based inhibitor of aldolase was identified, and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrich syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.     

Starvation-induced lysosomal degradation of aldolase B requires glutamine 111 in a signal sequence for chaperone-mediated transport

Aldolase B is an abundant cytosolic protein found in all eukaryotic cells. Like many glycolytic enzymes, this protein was sequestered into lysosomes for degradation during nutrient starvation. We report here that the degradation of recombinant aldolase B was enhanced two-fold when rat and human hepatoma cells were starved for amino acid and serum. In addition, starvation-induced degradation of aldolase B was inhibited by chloroquine, an inhibitor of lysosomal proteinases and by 3-methyladenine, an inhibitor of autophagy. Aldolase B has three lysosomal targeting motifs (Q(12)KKEL, Q(58)FREL, and IKLDQ(111)) that have been proposed to interact with hsc73 thereby initiating its transport into lysosomes. In this study, we have mutated the essential glutamine residues in each of these hsc73-binding motifs in order to evaluate their roles in the lysosomal degradation of aldolase B during starvation. We have found that when glutamines 12 or 58 are mutated to asparagines enhanced degradation of aldolase B proceeded normally. However, when glutamine 111 was mutated to an asparagine or a threonine, starvation-induced degradation was completely suppressed. These mutations did not appear to alter the tertiary structure of aldolase B since enzymatic activity was not affected. Our results suggest that starvation-induced lysosomal degradation of aldolase B requires both autophagy and glutamine 111. We discuss the possible roles for autophagy and hsc73-mediated transport in the lysosomal sequestration of aldolase B.     

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.     

A novel anti-aldolase C antibody specifically interacts with residues 85–102 of the protein

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85–102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C’s functions in the brain.     

A novel anti-aldolase C antibody specifically interacts with residues 85–102 of the protein

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85–102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C’s functions in the brain.     

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA₃ promoted an increase in aldolase accumulation when 0.1 M GA₃ was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 M GA₃, FBPA activity increased very little by 0.1 M GA₃ treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H⁺-ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA₃. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA₃-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.     

Negative charge correlates with neural expression in vertebrate aldolase isozymes

Electrophoretic studies suggest that negatively charged neural proteins are a general feature of jawed vertebrates. In an apparent example of this, teleost fish express three aldolase isozymes, one of which is expressed predominantly in neural tissues and is more negatively charged than its more generally expressed paralogues. We characterized three aldolase isozymes from a single species of teleost fish, zebrafish (Danio rerio). These sequences indicated that the correlation of net negative charge and neural expression suggested in other species by gel electrophoresis was supported by sequence analysis. When aldolase sequences from the databases were included in phylogenetic analyses, the negative charge/neural expression phenomenon was observed across the gnathostome vertebrate sequences examined. We found no evidence for a period of positive Darwinian selection resulting in an accumulation of negatively charged amino acids during the evolution of the neural aldolase isozymes. This is likely attributable, however, to limitations associated with the age of the duplication responsible for the neural isozyme and the reconstruction of ancestral sequences.     

Compartmentation of Aldolase Isoforms in Maize Leaves

The leaves of maize seedlings contain two principal isozymesof fructose 1,6-bisphosphate aldolase (E.C. 4.1.2.13 [EC] ), one chloroplasticand one cytosolic (Gasperini and Pupillo, 1982). Mesophyll protoplastswere separated from bundle sheath (BS) strands of both light-grownand dark-grown maize leaves. Aldolase isozymes were separatedfrom extracts of chloroplasts, etioplasts, protoplasts and BSstrands by column isoelectric focusing. The major isozyme ofgreen leaves (pI 4.2) was exclusively in BS chloroplasts, andthere was no evidence of other isozymes occurring in BS tissue.The cytosolic isozyme (pI 6.7) was present in protoplasts ofmesophyll cells, where it may limit the synthesis of hexose-phosphates(estimated activity of 9.4 µmol h^–1 g^–1 fr.wt.) together with lower activities of an acidic form (pI 4.6).Etiolated leaves contained significant amounts of the pI 6.7isozyme in both mesophyll and BS cells, but also minor activitiesof one or more acidic forms with pI values of 4.4–4.7(average pI 4.6) which appear to be located partly in BS etioplasts.The main developmental events for maize leaf aldolase afterillumination were a moderate decrease of cytosolic isozyme (pI6.7) which disappears from the BS within hours and a large,gradual increase of the BS plastid isozyme (pI 4.2). The isoformwith a pI 4.6 also increased rapidly to a low, steady activityin greening mesophyll protoplasts. Key words: C₄, fructose 1,6-bisphosphate, aldolase, Zea mays