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PubMed1970

Chloroplast aldolase is controlled by a nuclear gene.

Variant chloroplast fructose 1,6-diphosphate aldolases were found in Pisum sativum when 10 commercial varieties were examined for electrophoretically distinct species of chloroplast triose phosphate isomerase, phosphoglyceric acid kinase, glyceraldehyde 3-phosphate dehydrogenase, and aldolase. When reciprocal crosses are made, both aldolases appear in individuals in the F(1) generation. Backcrossing gives offspring having aldolases characteristic of the homozygous or of the heterozygous parent; the inheritance is therefore not maternal but Mendelian. Clearly this chloroplast reductive pentose phosphate cycle enzyme is under nuclear gene control in P. sativum.

Cell Nucleus↗
PubMed2004

Structure of aldolase from Thermus thermophilus HB8 showing the contribution of oligomeric state to thermostability.

2-Deoxyribose-5-phosphate aldolase catalyzes a reversible aldol condensation of two aldehydes via formation of a covalent Schiff-base intermediate at the active lysine residue. The crystal structure of 2-deoxyribose-5-phosphate aldolase from Thermus thermophilus HB8 has been determined with and without the substrate at atomic resolution. This enzyme, which has a unique homotetramer structure, has been compared with the previously reported crystal structures of two orthologues from Escherichia coli and Aeropyrum pernix. In contrast to the similar alpha/beta-barrel fold of the monomers, substantial quaternary structural differences are observed between these three enzymes. Further comparison of the subunit-subunit interface areas of these aldolases showed a clear positive correlation between the interface area and the living temperature of the source organism. From these results, it is concluded that the oligomeric state of 2-deoxyribose-5-phosphate aldolase is important for the thermostability and not for the catalytic function.

Aeropyrum↗
PubMed1977

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus.

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.

Animals↗
PubMed1980

The aldolase-substrate intermediates and their interaction with glyceraldehyde-3-phosphate dehydrogenase in a reconstructed glycolytic system.

The relative concentration of the aldolase x fructose-bisphosphate and of the aldolase x dihydroxy-acetone-phosphate complexes is regulated, in the steady state, by the nature of the accompanying glycolytic enzymes. Particularly in the presence of triose phosphate isomerase, the aldolase x dihydroxyactone-phosphate complexes are largely prevalent. This situation is very likely to hold in rabbit muscle in vivo. Aldolase and gyceraldehyde-3-phosphate dehydrogenase slowly form a complex; however, no evidence has been found for the direct transfer of glyceraldehyde 3-phosphate between the two enzymes.

Animals↗
PubMed1985

Affinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine.

Aldolase contains one tight binding site and one weak binding site per subunit for ATP [Kasprzak, A. and Kochman, M. (1980) Eur. J. Biochem. 104, 443-450]. The reaction of the ATP analog 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit aldolase A results in linear inactivation of enzyme with respect to covalent linkage of fluorescent label. The enzyme is completely protected against modification in the presence of saturating covalent binding (k2 = 0.033 min-1) is preceded by a fast reversible binding step (Ki = 6.8 mM). Chemical modification of aldolase leads to formation of stable N epsilon (4-carboxybenzenesulfonyl-lysine (Cbs-Lys) and O-(4-carboxybenzenesulfonyl-tyrosine (Cbs-Tyr) derivatives. Almost all Cbs-Lys was found in the N-terminal CNBr peptide (CN-1), whereas Cbs-Tyr was present both in the N-terminal (CN-1) and C-terminal (CN-2) peptide. From carboxypeptidase digestion and tryptic peptide analysis, Cbs-Lys was localized in position 107, a small part of Cbs-Tyr was detected in position 84, and the majority of Cbs-Tyr was found in the C-terminal position Tyr-363. We conclude that the covalent binding of the ATP analog occurs at the mononucleotide tight-binding site of aldolase and is associated with modification of Lys-107 and Tyr-363. This conclusion is based on the measurements of enzymatic activity loss as a function of ATP analog incorporation as well as on previous data. It is postulated that Lys-107, which is the C-6 phosphate binding site for fructose-1,6-P2, is in close proximity to the functionally important Tyr-363. The rather small extent of modification of Tyr-84 (0.15 mol/subunit), is due either to nonspecific protein modification or labeling of the weak mononucleotide binding site.

Adenosine↗
PubMed1987

A new human species of aldolase A mRNA from fibroblasts.

A full-length cDNA aldolase A clone was isolated from a human fibroblast cDNA library and completely sequenced. Excluding the poly(A) tail, the clone covers 1095 base pairs (bp) of the coding region, plus 199 bp downstream for the termination codon and 146 bp upstream for the initiation codon, within a total of 1440 bp. Primer extension experiments performed with human cultured fibroblast mRNA indicate an elongated product of a further 40 bp. These results evaluated together with those obtained in a concurrent study concerning aldolase A mRNA isolated from human liver are direct evidence of aldolase A mRNA multiplicity in man. The data also suggest the existence in mammals of three different classes of aldolase A mRNA, which would account for tissue specificity and resurgence of foetal expression in tumors.

Amino Acid Sequence↗
PubMed1990

Characterization of the transcription-initiation site and of the promoter region within the 5' flanking region of the human aldolase C gene.

Several aldolase C clones from a human genomic library have been identified using a mouse aldolase C cDNA as a hybridization probe. The most complete fragment of the clones identified is 14 kb long and contains the complete aldolase C gene. The nucleotide sequence analysis of more than 5 kb includes the intron/exon organization structure of the gene and the 3' and 5' flanking regions. Although no human cDNA is yet available, a canonical polyadenylation signal at the 3' end of the gene indicates the proximity of the poly(A) addition site. We have analyzed the 5' noncoding region by S1 mapping and primer-extension experiments. The transcription-initiation sites for the human aldolase C gene in brain tissue was located about 1300 bp upstream from the methionine initiation codon. Preliminary functional assays of the promoter by transfection into rat glioma cells have indicated that promoter elements lie between positions -161 and -416 from the start point of transcription.

Amino Acid Sequence↗
PubMed1991

An additional promoter functions in the human aldolase A gene, but not in rat.

The aldolase A gene was isolated from a human DNA library, mapped and sequenced. This gene comprises 12 exons and spans 6.5 kb. From the genomic DNA sequence and from the previous sequence analysis of the cDNA, it was revealed that the first exon L1 and the second exon encode the 5' non-coding sequence of mRNA L1, while the third and forth exons (corresponding to exons M and L2) encode different mRNA, mRNA M and L2, respectively; the following eight exons (exons 5-12) are shared commonly by all the mRNA species. These results indicate that the mRNA species are generated from a single aldolase A gene from one of exons L1, M or L2, in addition to exons 5-12, and also that the usage of a leader exon is similar but clearly distinct from that of rat aldolase A gene which we analyzed [Joh, K., Arai, Y., Mukai, T. & Hori, K. (1986) J. Mol. Biol. 190, 401-410]. By comparing the promoter regions in the human and rat aldolase A genes, we found similar sequences in the rat genome corresponding to those of the human L1, M and L2 promoter. We could not, however, detect any transcripts starting from sequences corresponding to the human L1 promoter in the rat genome, although the products corresponding to human M and L2 were detected. Thus, we conclude that the L1 promoter was either acquired by the human genome or deleted from the rat genome after human and rat diverged during evolution.

Amino Acid Sequence↗
PubMed1994

Position-independent, high-level, and correct regional expression of the rat aldolase C gene in the central nervous system of transgenic mice.

Aldolase C is mainly expressed in the central nervous system (CNS). To clarify the regulatory mechanisms for the CNS-specific expression, transgenic mice were created using two constructs of the rat aldolase C gene. A fusion gene comprising the 5' regulatory region of the aldolase C gene was expressed in a CNS-specific manner. However, the expression levels and the cellular localization of the gene varied among transgenic mice. The other construct, including both 5' and 3' regulatory regions of the gene, showed position-independent and high-level expression as well as the correct regional distribution in the CNS. These results indicate that the 13-kb sequence of the rat aldolase C gene contains sufficient information for faithful expression of the gene.

Animals↗
PubMed1982

Molecular studies of liver aldolase B in hereditary fructose intolerance using blotting and immunological techniques.

Hereditary fructose intolerance is due to a deficiency of liver aldolase (aldolase B). Little is known about its molecular mechanisms. We have tried to demonstrate the presence of the molecule and have explored the possibility of genetic heterogeneity. Liver samples from fifteen cases of hereditary fructose intolerance due to aldolase B deficiency were studied by various electrophoretic techniques. After electrophoresis on polyacrylamide gels, proteins were electrophoretically transferred on to nitrocellulose filters. They were treated with specific antialdolase B antibodies, and then with radioiodinated protein A, followed by autoradiography. Investigations included: (a) sodium dodecyl sulphate electrophoresis, in order to detect the presence of immunologically reactive molecules and to estimate the subunit size; (b) attempts to discover charge anomalies of the native molecule and of its subunits, by the use of: Isoelectric focusing of the native enzyme. Isoelectric focusing and non-equilibrium pH gradient electrophoresis (NEPHGE) after dissociation in urea. The major results were the following: (1) In all cases a cross-reacting material was found, with a molecular subunit size of 38000, indistinguishable from that of controls. (2) Evidence for molecular heterogeneity of the disease was provided by two types of data: amount of apparent immunologically reactive protein, which varied from less than 3% to 100% of that of controls; and charge data, aldolase B from seven patients showing an increased negative charge and from one patient a normal charge.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1984

Saccharomyces cerevisiae aldolase mutants.

Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo.

Fructose-Bisphosphate Aldolase↗
PubMed1962

Some characteristics of a purified heat-stable aldolase.

Thompson, P. J. (University of Nebraska, Lincoln) and T. L. Thompson. Some characteristics of a purified heat-stable aldolase. J. Bacteriol. 84:694-700. 1962-Aldolase from a thermophilic strain of bacteria was obtained in a state of high purity. Heat studies of purified aldolases from cells cultivated at 45 and 65 C showed them equally stable at 70 C for 1 hr. Metal-ion and chelate studies indicated that thermal aldolase is metal ion-independent. Carboxypeptidase did not alter activity or specificity. The enzyme was specific for fructose-1,6-diphosphate. Hydrazine was found inhibitory in the assay procedure. The inhibition was independent of pH over the range of H(+) concentrations tested and was reversed by dialysis against water.

Aldehyde-Lyases↗
PubMed1976

Differential effects of pyridoxal 5'-phosphate on lysine residues in rabbit and sturgeon muscle aldolases.

Pyridoxal 5'-phosphate (PLP) in aqueous solutions can form a Schiff base complex with 14 and 16 lysine residues of rabbit and sturgeon muscle aldolases (EC 4.1.2.13), respectively. Although the mechanism of their interaction with PLP should be the same, these residues can be differentiated into three families on the basis of their inhibition constant Ki and rate constant k. The lysine residues of one of these families do not react with PLP in the presence of the substrates. Therefore, they are assumed to be part of the active center. In the sturgeon muscle aldolase, 3.7 substrate protected lysine residues are present. Rabbit aldolase, although tetrameric, contains only 2.8 substrate protected lysine residues. This suggests that one active center of this enzyme may be 'buried'. Structural studies showed the following sequence around the substrate protected lysine residues, in the rabbit aldolase: Gly-(Gly2, Val3)-Pyridoxyl Lys-Ile-Asp-Lys.

Amino Acids↗
PubMed2001

PCR primers for an aldolase-B intron in acanthopterygian fishes.

BACKGROUND: Nuclear DNA sequences provide genetic information that complements studies using mitochondrial DNA. Some 'universal' primer sets have been developed that target introns within protein-coding loci, but many simultaneously amplify introns from paralogous loci. Refining existing primer sets to target a single locus could circumvent this problem. RESULTS: Aldolase intron 'G' was amplified from four fish species using previously described primer sets that target several loci indiscriminately. Phylogenetic analyses were used to group these fragments and other full-length aldolase proteins from teleost fishes into orthologous clades and a primer set was designed to target specifically an intron within the aldolase-B locus in acanthopterygian fishes. DNA amplifications were tried in a variety of acanthopterygian fishes and amplification products, identifiable as aldolase-B intron 'G', were observed in all atherinomorph and percomorph taxa examined. Sequence variation within this locus was found within and among several species examined. CONCLUSIONS: Using 'universal' primer sets coupled with phylogenetic analyses it was possible to develop a genetic assay to target a specific locus in a variety of fish taxa. Sequence variation was observed within and among species suggesting that this targeted assay might facilitate interspecific and intraspecific comparisons.

Amino Acid Sequence↗
PubMed1994

The cloning of zebrin II reveals its identity with aldolase C.

The sagittal organization of the mammalian cerebellum can be observed at the anatomical, physiological and biochemical level. Previous screening of monoclonal antibodies produced in our laboratory has identified two intracellular antigens, zebrin I and II, that occur exclusively in adult cerebellar Purkinje cells. As their name suggests, the zebrin antibody staining of the Purkinje cell population is not uniform. Rather, zebrin-positive Purkinje cells are organized in stripes or bands that run from anterior to posterior across most of the cerebellum; interposed between the zebrin-positive cells are bands of Purkinje cells that are zebrin-negative. Comparison of the position of the antigenic bands with the anatomy of afferent projections suggests that the bands are congruent with the basic developmental and functional 'compartments' of the cerebellum. We report the isolation of cDNA clones of the 36 x 10(3) M(r) antigen, zebrin II, by screening of a mouse cerebellum cDNA expression library. Sequence analysis reveals a 98% identity between our clone and the glycolytic isozyme, aldolase C. In order to more rigorously demonstrate the identity of the two proteins, we stained adult cerebellum with an independent monoclonal antibody raised against aldolase C. Anti-aldolase staining occurs in a previously unreported pattern of sagittal bands of Purkinje cells; the pattern is identical to that revealed by the zebrin II monoclonal. Further, in situ hybridization of antisense aldolase C riboprobe shows that the accumulation of zebrin II/aldolase C mRNA corresponds to the pattern of the zebrin antigen in Purkinje cells. Zebrin II/aldolase C gene expression is thus regulated at the level of transcription (or mRNA stability). In light of previous work that has demonstrated the cell-autonomous and developmentally regimented expression of zebrin II, further studies of the regulation of this gene may lead to insights about the determination of cerebellar compartmentation.

Amino Acid Sequence↗
PubMed2000

Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase gene cluster from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus, and phylogeny of the fructose 1,6-bisphosphate aldolase encoded by cbbA in the cluster.

Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila). cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria. cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins. When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE. However, the cell extract had slightly higher activity than a cell extract of E. coli without cbbA. Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H. thermoluteolus was in class IIA. Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions. The activities of the two enzymes were regulated independently.

Amino Acid Sequence↗
PubMedDate not supplied

Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces.

The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25 degrees C the tetramer-dimer dissociation constants for each single-mutant enzyme are similar, about 10(-6) M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramer-monomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10(-15) M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10(-28) M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.

Centrifugation, Density Gradient↗
PubMed2004

Molecular, topographic, and functional organization of the cerebellar cortex: a study with combined aldolase C and olivocerebellar labeling.

Aldolase C (zebrin) expression in Purkinje cells reveals stripe-shaped compartments in the cerebellar cortex. However, it is not clear how these compartments are related to cerebellar functional localization. Therefore, we identified olivocerebellar projections to aldolase C compartments by labeling climbing fibers with biotinylated dextran injected into various small areas within the inferior olive in rats. Specific rostral and caudal aldolase C compartments were linked in an orderly manner by common olivocerebellar projection across the rostrocaudal boundary on lobule VIc-crus Ib. Based on the localization of the olivary origins of projection to similar compartments, the compartments and olivocerebellar projections could be sorted into five groups: group I, positive compartments extending from the posterior lobe to the anterior lobe innervated by the principal olive and some neighboring areas; group II, positive compartments localized within the posterior lobe innervated by several medial subnuclei; group III, vermal and central negative compartments innervated by the centrocaudal medial accessory olive; group IV, negative and lightly positive compartments in the hemisphere and the rostral and caudal pars intermedia innervated by the dorsal accessory olive and some neighboring areas; group V, the flocculus and nodulus. The olivocerebellar topography within each group was simple and suggests an "orientation axis" within the concerned parts of the inferior olive. Furthermore, parts of the inferior olive in each group receive specific afferent inputs, indicating a close relationship between aldolase C compartments and functional localization. Thus, the five-group scheme we propose here may integrate the molecular, topographic, and functional organization of the cerebellum.

Animals↗