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PubMed1983

Radioimmunoassay of aldolase A. Determination of normal serum levels and increased serum concentration in cancer patients.

A radioimmunoassay specific for human aldolase A subunits was used to measure human aldolase A (ALD-A) in human serum. The double antibody competitive inhibition radioimmunoassay technique used radioiodinated purified ALD-A as ligand, chicken antisera specific for human ALD-A and rabbit antichicken IgG. The serum levels of ALD-A in 42 normal healthy subjects ranged from 130 to 210 ng/ml (mean average, 171 +/- 39 ng/ml). In 177 hospitalized patients without cancer, muscle diseases, or hemolytic anemia, the ALD-A serum levels ranged from 125 to 220 ng/ml. In contrast, 82% of 260 patients with various types of malignancy had ALD-A serum concentrations above the normal range. The CEA levels increased only 44% of the sera of 80 patients with cancer of the digestive tract, whereas the ALD-A levels were increased in 86% of the patients. The AFP levels were greater than 100 ng/ml in only 70% of the sera of 33 liver cell carcinoma patients, whereas the ALD-A levels were increased in 94% of these sera. The measurement of serum ALD-A by radioimmunoassay may be a valuable adjunct in the clinical diagnosis of certain cancer patients.

Adolescent↗
PubMed1995

Molecular basis of hereditary fructose intolerance: mutations and polymorphisms in the human aldolase B gene.

Mutations in the human aldolase B gene that result in hereditary fructose intolerance have been characterized extensively. Although the majority of subjects have been from northern Europe, subjects from other geographical regions and ethnic groups have been identified. At present 21 mutations have been reported; 15 of these are single base substitutions, resulting in nine amino acid replacements, four nonsense codons, and two putative splicing defects. Two large deletions, two four-base deletions, a single-base deletion, and a seven-base deletion/one-base insertion have been found. This last mutation leads to a defect in splicing and it is likely that one of the small deletions does as well. Regions of the enzyme where mutations have been observed recurrently are encoded by exons 5 and 9. Indeed, the three most common mutations are found in these exons. Two of these prevalent HFI mutations arose from a common ancestor and spread throughout the population by genetic drift. This finding was based on linkage to two sequence polymorphisms, which are among very few informative polymorphic markers that have been identified within the aldolase B gene. Because of the prevalence of a few HFI alleles, and the recent advances in molecular methods for identifying and screening for mutation, the diagnosis of HFI by molecular screening methods should become routine. These molecular diagnostic methods will be extremely beneficial for this often difficult to diagnose and sometimes fatal disease.

Chromosome Mapping↗
PubMed1990

Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.

Amino Acid Sequence↗
PubMed1991

In vitro translation of Plasmodium falciparum aldolase is not initiated at an unusual site.

It has been proposed recently that translation of fructose-1,6-diphosphate aldolase of the malaria parasite Plasmodium falciparum is initiated at a UAG codon, both in the parasite and in a rabbit reticulocyte cell-free translation system. We have introduced mutations around that UAG codon and find that cell-free expression of a construct encoding an AUG in this position results in a slightly larger translation product. The translation product of the construct encoding the UAG codon is of the same apparent molecular weight as the products obtained from two other constructs; one in which the UAG is replaced by AAG, and one in which nucleotides upstream from a second AUG codon are deleted. Thus we show that translation is not initiated at the UAG and conclude that synthesis of aldolase in the parasite starts at an AUG, provided after splicing of pre-mRNA.

Animals↗
PubMed1978

Localization of pyruvate kinase isozymes in bovine kidney and comparison of these patterns with those of lactate dehydrogenases and aldolases.

Electrophoretic and immunofluorescence analysis were used to study the distribution of pyruvate kinase isozymes in the bovine kidney. Electrophoretic analysis demonstrated the presence of large amounts of K4 plus small amounts of K-M hybrids in cortical, medullary, and papillary sections cut from the kidney. Nearly all of the K-L hybrids seen in whole kidney extracts were found in cortical sections. Immunofluorescence of frozen sections revealed the presence of type L subunits in the tubules but the complete absence of this subunit type in flomeruli. Glomeruli do contain large quantities of pyruvate kinase isozymes, probably K4 and K-M hybrids, that cross-react with antibodies produced against type M pyruvate kinase. Type L-containing forms of pyruvate kinase and aldolase type B both appear to be found in cell types thought to be capable of catalyzing of gluconeogenesis, while type K pyruvate kinase and type A aldolase are found in predominantly glycolytic cell types of the kidney. Lactate dehydrogenase isozymic patterns appear to be less closely correlated with glycolytic versus gluconeogenic functions of the kidney but may be determined more directly by other metabolic functions.

Animals↗
PubMed1979

Fructose-1,6-bisphosphate aldolase from Vibrio marinus, a psychrophilic marine bacterium.

Fructose-1,6-bisphosphate aldolase (Fru-P2A) from a psychrophilic marine bacterium was found to be Class II aldolase based on activation by K+, activation by divalent cations, inactivation by EDTA, low molecular weight, and similar values for Km, Vmax, and Arrhenius activation energy. This enzyme was not markedly different in amino acid composition from the enzymes from mesophilic and thermophilic organisms, yet it has unusual thermal properties.

Amino Acids↗
PubMed1994

Dietary and hormonal regulation of aldolase B gene transcription in rat liver.

In the liver of the fasted rat, the aldolase B (AldB) mRNA level decreased to about half of that of the control rat. When the control rat was refed the glucose-rich diet, the AldB mRNA level increased about six to seven times more than in the fasted rat. This increase was shown as the activation of the AldB gene transcription by a nuclear run-on assay. To understand the causal factor(s) for this activation, the relationship between the AldB mRNA level in the liver and the plasma concentrations of hormones, which are known as major regulators of carbohydrate metabolism during fasting and refeeding, was investigated. The plasma insulin level in the rat which was refed the glucose-rich diet increased in parallel to AldB mRNA level, while the plasma glucagon level decreased reciprocally to it. The relationship of the plasma corticosterone level to the AldB mRNA level was not obvious. To directly confirm the effects of these hormones on AldB gene transcription in the liver, the responses of AldB gene in the primary cultured hepatocytes to these hormones were examined. Insulin and dexamethasone were effective to activate AldB gene, while glucagon and thyroxine were suppressive. Thyroxine did not extinguish the effects of insulin and dexamethasone, but glucagon canceled them. Thus, it is probable that in vivo these hormones synergistically regulate the AldB gene transcription. In vitro transcription analysis of two AldB promoter constructs suggested that the proximal half of the AldB promoter (up to -92 bp from the transcription start site) is, at least in part, involved for this induction, and the distal half which contains liver-specific elements (-93 to -202 bp) is not involved. The possible explanation for the dietary regulation of aldolase B gene transcription in the liver is discussed.

Animals↗
PubMed1998

Purification and characterization of cytosolic fructose-1, 6-bisphosphate aldolase from endosperm of germinated castor oil seeds.

Cytosolic fructose-1,6-bisphosphate (FBP) aldolase (ALDc) from the endosperm of 4- to 5-day germinated castor oil seeds (COS) has been purified 83-fold to electrophoretic homogeneity and a final specific activity of 2.5 micromol FBP cleaved/min/mg protein. SDS-PAGE and denaturing isoelectric focusing of the final preparation revealed a single protein-staining band of 40 kDa and pI value 7.2. The native Mr was determined by gel-filtration chromatography and multiangle laser light scattering to be 160-175 kDa, indicating that the enzyme is homotetrameric. The enzyme (a) is a class I aldolase, since EDTA or Mg2+ had no effect on its activity; and (b) was relatively heat stable and had an activation energy of 100 kJ/mol. It exhibited a broad pH-activity optima of 7.2, a relatively high affinity for FBP (Km = 0.16 microM), and a forward:reverse Vmax ratio of 0.77. Rabbit anti-(COS ALDc) antibodies cross-reacted with COS ALDc, but not with the corresponding plastidic isozyme. Time-course studies revealed that (a) the increase in total ALD activity that occurs during COS development and early germination coincides with an increase in ALDc concentration and (b) the latter stages of COS maturation and germination are accompanied by marked reductions in ALD activity and ALDc concentration. The most significant elevation in ALDc concentration occurred over the first 4 days of germination when COS initiates the gluconeogenic conversion of P-enolpyruvate and triose-P, derived from reserve triacylglycerols, into the sucrose required to support early seedling growth.

Amino Acids↗
PubMed1996

The aldolase A promoter in proliferating rat thymocytes is regulated by a cluster of SP1 sites and a distal modulator.

In mitogen-stimulated rat thymocytes the activities and mRNA levels of aldolase A increase remarkably during proliferation pointing to a transcriptional regulation of this enzyme. By DNAse I footprinting and mobility shift competition assays five binding sites for the activating transcription factor Sp1 and one site for an AP-1 like nuclear factor could be identified in the core activating region of the proximal aldolase AH1 promoter downstream of -400. Transfection data and differences found in nuclear protein binding of resting and proliferating cells to DNA sites suggest that Sp1 is an integral part of the mechanism by which the AH1 promoter achieves high level transcription during proliferation. Moreover we demonstrate that an element between positions -1066/-731 significantly attenuates the AH1 promoter driven transcription as well as transcription regulated by the heterologous SV40 promoter. From this effect a functional linkage between the distal muscle-restricted M1 promoter and the active AH1 promoter can be suggested.

Animals↗
PubMed1997

Identification of the human aldolase A gene as the first induced target for the TR2 orphan receptor, a member of the steroid hormone receptor superfamily.

The human TR2 orphan receptor (TR2) is a member of the steroid/thyroid hormone receptor superfamily that regulates the transcription of complex gene networks and subsequently controls diverse aspects of growth, development, and differentiation. In the present study, we have found that the TR2 is one of the M1 site (nucleotide numbers 2017-2034, 5'-AAAAGGGCAGGGGTCATT-3') binding proteins of the muscle-specific pM promoter in the human aldolase A gene. Electrophoretic mobility shift assay (EMSA) showed a specific binding with high affinity (dissociation constant = 4.6 nM) between the TR2 and the M1 element. Circular permutation assay revealed a localized DNA flexibility induced by the TR2 binding, and the bend angle was estimated to be 73 +/- 2 degrees. Furthermore, a dual-luciferase reporter gene assay demonstrated that the TR2 may enhance the expression of luciferase activities via the wild-type M1 site but not the mutant M1 element in human QM7 muscle myoblasts. In conclusion, our data represent the first case of demonstrating that the TR2 may serve as a transcriptional inducer in muscle-specific aldolase A gene expression.

Base Sequence↗
PubMed1997

Sequence requirement for replication initiation at the rat aldolase B locus implicated in its functional correlation with transcriptional regulation.

Transcription promoter of the aldolase B gene was previously shown to be centered on an initiation region of DNA replication in rat hepatoma cells in vivo. Here, we defined an essential region required for replication in a plasmid form upon transfection. Deletion analyses around the origin region revealed that the proximal 200 bp promoter was necessary, but not sufficient for replication as flanking sequence restored replication activity. Therefore, the 200 bp region seemed to cooperate with the flanking sequence to play an important role in replication. Electrophoretic mobility shift assays using nuclear extracts from synchronously growing hepatoma cells showed that some protein factors bound to this region in a cell cycle-regulated manner. Since transcription of the aldolase B gene is repressed in the hepatoma cells, the cell cycle-regulated protein-binding is considered to be involved in regulation of replication initiation.

Animals↗
PubMed1999

Conserved residues in the mechanism of the E. coli Class II FBP-aldolase.

The two classes of fructose-1,6-bisphosphate aldolase both catalyse the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The Class I aldolases use Schiff base formation as part of their catalytic mechanism, whereas the Class II enzymes are zinc-containing metalloproteins. The mechanism of the Class II enzymes is less well understood than their Class I counterparts. We have combined sequence alignments of the Class II family of enzymes with examination of the crystal structure of the enzyme to highlight potentially important aspartate and asparagine residues in the enzyme mechanism. Asp109, Asp144, Asp288, Asp290, Asp329 and Asn286 were targeted for site-directed mutagenesis and the resulting proteins purified and characterised by steady-state kinetics using either a coupled assay system to study the overall cleavage reaction or using the hexacyanoferrate (III) oxidation of the enzyme bound intermediate carbanion to investigate partial reactions. The results showed only minor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and Asp329 mutants, suggesting that these residues play only minor or indirect roles in catalysis. By contrast, mutation of Asp109 or Asn286 caused 3000-fold and 8000-fold decreases in the kcat of the reaction, respectively. Coupled with the kinetics measured for the partial reactions the results clearly demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate. Fourier transform infra-red spectroscopy of the wild-type and mutant enzymes has further delineated the role of Asp109 as being critically involved in the polarisation of the carbonyl group of glyceraldehyde 3-phosphate.

Escherichia coli↗
PubMed2002

A functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.

Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the beta5-alpha7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-(2)H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that flexibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK(a) to a value suitable for its role in proton abstraction. These results highlight the need for further studies of the dynamics of the enzyme in order to fully understand the complexities of loop closure and catalysis in this enzyme.

Amino Acid Substitution↗
PubMed1995

High-level expression of Trypanosoma brucei fructose-1,6-bisphosphate aldolase in Escherichia coli and purification of the enzyme.

A procedure has been developed for high-level expression of Trypanosoma brucei fructose-bisphosphate aldolase in Escherichia coli. Therefore, a specific restriction site was introduced by mutagenesis at the front of the gene, enabling its ligation in an expression plasmid, immediately downstream of the regulatory sequences. Growth conditions were established for production of high amounts of soluble and active enzyme. Aldolase was purified to near-homogeneity from the soluble fraction of the bacterial lysate by nuclease treatment, differential precipitation steps, and passage over a CM-Sepharose column. From a 1-liter culture of E. coli cells, 60-120 mg of purified protein that is essentially indistinguishable in physicochemical and kinetic properties and in stability from the enzyme purified from trypanosomes grown in infected laboratory animals was reproducibly obtained.

Animals↗
PubMed1988

Chemical modification of the actin binding site of rabbit muscle aldolase by diethylpyrocarbonate.

To extend the available information on the significance of the interactions between glycolytic enzymes and the actin component of the cellular ultrastructure, investigations into the compositional characteristics of the actin binding site on one of the major glycolytic enzymes, aldolase, have been undertaken. As the electrostatic nature of the association has been previously reported indicative of a cationic region on the enzyme involved in the binding, these studies have investigated the possibility of the involvement of histidine residues in this binding region. By the use of the histidine specific reagent, diethylpyrocarbonate, we have been able to establish a difference in nature of an actin binding domain and the active site domain which does contain an essential histidine. The results have been discussed in relation to the significance of this finding with respect to the binding of aldolase to subcellular structure.

Actins↗
PubMed1987

Mapping of a restriction fragment length polymorphism within the human aldolase B gene.

Peripheral blood DNA was hybridized to the full-length cDNA and the cloned structural gene of human aldolase B. With PvuII endonuclease a restriction fragment length polymorphism was detected that was present in the heterozygous state in about 21% of the individuals tested. A map of the human aldolase gene was constructed for the two groups of individuals found to produce different fragments after PvuII digestion. This allowed the localization of the polymorphic site within the gene, which was found to be due to the loss of a PvuII site in the last intron upstream from the 3' end. This polymorphism may be used as a genetic marker to study individuals affected by hereditary fructose intolerance.

Chromosome Mapping↗
PubMed1987

Molecular gene mapping of human aldolase A (ALDOA) gene to chromosome 16.

Mapping of human aldolase A (ALDOA) gene was performed by molecular hybridization techniques using a panel of human-mouse cell hybrids and sorted fractions of human metaphase chromosomes besides in situ hybridization. For the purpose, three kinds of DNA probes derived from the coding region (probe-1), the 3' noncoding region (probe-2), and the coding and 3' noncoding regions (probe-3) of human aldolase A cDNA clone, pHAAL116-3, were selectively employed. The results of RNA and DNA blot analyses indicated that the human ALDOA gene is located on chromosome 16. The in situ hybridization experiment also indicated that the ALDOA gene was localized to 16q22-q24.

Animals↗
PubMed1995

Aldolase C/zebrin II and the regionalization of the cerebellum.

The cerebellum is comprised of multiple bands of cells, each with characteristic afferent and efferent projections, and patterns of gene expression. The most studied example of a striped pattern of expression is the antigen recognized by monoclonal antibody antizebrin II. Zebrin II is expressed by subsets of Purkinje cells that form an array of parasagittal bands that extend rostrocaudally throughout the cerebellar cortex, separated by similar bands of Purkinje cells that do not express zebrin II. Recent cloning studies have revealed that the zebrin II antigen is the respiratory isoenzyme aldolase C. This article reviews the cellular and molecular compartmentation of the cerebellum together with the molecular biology of the aldolase C gene, and speculates on possible reasons for a striped pattern of expression.

Brain Chemistry↗