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PubMed1975

Isozyme pattern of fructose diphosphate aldolase during hepatocarcinogenesis induced by 2-acetylaminofluorene in rat liver.

The biosynthesis of aldolase A and B subunits has been studied in rat liver during the administration of carcinogen AAF4. Transition from a predominance of aldolase B to A was observed during carcinogenesis in rat liver. Changes in isozymic pattern and FDP to F-1-P cleavage activity ratio were observed before histological alterations typical of hepatoma could be detected. Our data support the hypothesis of dedifferentiation during hepatocarcinogenesis which in an early stage results in switching on of the gene for aldolase A with simultaneous continuation of biosynthesis of aldolase B within single cells.

2-Acetylaminofluorene↗

PubMedDate not supplied

Brownian dynamics of interactions between aldolase mutants and F-actin.

Previous Brownian dynamics (BD) simulations (Ouporov IG, Knull HR and Thomasson KA 1999. Biophys. J. 76: 17-27) of complex formation between rabbit aldolase and F-actin have identified three lysine residues (K288, K293 and K341) on aldolase and acidic residues (DEDE) at the N-terminus of actin as important to binding. BD simulations of computer models of aldolase mutants with any of these lysine residues replaced by alanine show reduced binding energy; the greatest effect of a single substitution is for K341A, and replacement of all three lysines greatly reduces binding. BD simulations of wild-type rabbit aldolase vs altered F-actin show that binding is decreased if any one of the four N-terminal acidic residues is replaced by alanine and binding is greatly reduced if three or more of the N-terminal acidic residues are replaced; none of the four actin residues appear more critical for binding than the others.

Actins↗

PubMed1992

Reversible unfolding and refolding behavior of a monomeric aldolase from Staphylococcus aureus.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.

Enzyme Reactivators↗

PubMed2007

Modeling the interaction between aldolase and the thrombospondin-related anonymous protein, a key connection of the malaria parasite invasion machinery.

A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 A) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds.

Animals↗

PubMed1995

Expression of aldolase A steady-state mRNA is delayed relative to other muscle-specific genes during differentiation of chicken myoblasts.

Expression of several muscle-specific genes was monitored during chicken muscle development and myoblast differentiation in primary cultures. The individual patterns of expression for many muscle-specific genes are well documented in ovo and in other model systems of muscle development. However, comparison of aldolase A to other muscle-specific genes in one system has not been reported. Both sarcomeric and cytosolic genes important for the adult muscle fiber were examined in order to elucidate their timing of expression and its relationship to cell fusion. Steady-state mRNA expression was measured using RNase protection assays with cRNA probes generated from cDNA clones for muscle creatine kinase, fast skeletal troponin-T, embryonic myosin heavy chain, and aldolase A. Nonmuscle genes expressed largely in the embryo, aldolase C and beta-actin, were used as controls. The expression of all six genes revealed differences in temporal expression patterns between limb and axial muscle. The temporal expression patterns of all six genes were also monitored in primary myoblast cultures relative to myoblast fusion. In both axial and limb myoblast cultures most of the muscle-specific genes were expressed prior to fusion. During the differentiation of myoblasts to myotubes there was a biphasic pattern in the expression of the muscle-specific genes. The appearance of measurable mRNA was detected by 16 hr in culture, prior to appreciable fusion of the cells. During further differentiation the expression increased gradually and then more rapidly at 96 hr, once fusion was complete. Meanwhile, the nonmuscle embryonic gene expression declined only slightly. For one gene, aldolase A, expression was delayed relative to the other muscle-specific genes, both in the appearance of measurable mRNA and in the later rapid increase in mRNA.

Animals↗

PubMed2001

Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus.

Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-1, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49--54% identity, 76--81% homology). Taq FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40--50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent 21-amino-acid insertion sequence. The recombinant protein is thermostable for at least 45 min at 80 degrees C with little residual activity below 60 degrees C. Kinetic characterization at 70 degrees C, the optimal growth temperature for T. aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K(m) of 305 microM. The maximal specific activity (V(max)) is 46 U/mg at 70 degrees C.

Amino Acid Sequence↗

PubMed1988

Aldolase C is localized in neuroendocrine cells.

To elucidate the localization of the subunit C of aldolase (aldolase C) in peripheral neuroendocrine cells, we made an immunohistochemical study with monospecific antibodies against human aldolase C. Aldolase C was found to be localized in various types of neuroendocrine cells; in the pituitary gland, thyroid, pancreas, adrenal gland, bronchus, and gastrointestinal tract.

Fructose-Bisphosphate Aldolase↗

PubMed1981

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

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.

Animals↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed1977

Purification and properties of rabbit heart muscle aldolase.

Fructose diphosphate aldolase (D-fructose-1,6-biphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit heart has been purified and obtained in crystalline form. The preparations are homogeneous on the basis of disc gel electrophoresis and ultracentrifugation. The catalytic and the molecular properties indicate that this is aldolase A. A comparison was made between rabbit heart aldolase and the rabbit muscle enzyme. The sedimentation coefficient, energy of activation and Michaelis constant for Fru-1,6-P2 were found to be identical with the values obtained for the muscle enzyme. As in case of the muscle enzyme, heart aldolase was found to have a broad pH optimum, remarkable stability over a wide pH range, and the ability to form a Schiff base intermediate with dihydroxyacetone phosphate upon reduction with borohydride. Cleavage of the methionyl bonds with CNBr yields the same pattern as obtained with the muscle enzyme.

Amino Acids↗

PubMed1981

Studies on the structure of aldolase A from chicken muscle.

S-Carboxymethylated chicken muscle aldolase was treated with cyanogen bromide to cleave the 4 methionyl bonds per subunit. Five homogeneous fractions were obtained designated fragments I-V. Fragment I was derived from the N-terminus and fragment II from the C-terminus of the enzyme. Reduction of the enzyme with NaB3H4 in the presence of dihydroxyacetone phosphate decreases the enzymatic activity by 90%. Fragment III contained the Schiff base-forming lysine residue since more than 83% of the radioactivity introduced by NaB3H4 reduction of aldolase-dihydroxyacetone phosphate was found in this fraction. A tryptic peptide of 27 amino acid residues containing the substrate-binding site was isolated. The gross molecular structure of aldolase A from chicken muscle indicates a high degree of homology with mammalian muscle aldolases.

Alkylation↗

PubMed1985

Amino acid sequence of an invertebrate FBP aldolase (from Drosophila melanogaster).

The complete amino acid sequence of FBP aldolase from Drosophila melanogaster has been determined. The enzyme contains four identical subunits of 360 amino acid residues. The primary structure of the monomer was established using automated Edman degradation on fragments prepared by CNBr-cleavage, by partial acid cleavage at the unique Asp-Pro bond and by oxidative cleavage at the three tryptophan residues. Manual Edman-Chang degradation was used on smaller peptides obtained by digestion with Staphylococcus aureus V8 protease, trypsin or chymotrypsin. The primary structure of Drosophila aldolase exhibits very extensive homology with the sequence of rabbit muscle aldolase (71% identity), thus explaining the early observation that Drosophila and mammalian aldolases form active interspecies hybrid quaternary structures (Brenner-Holzach, O. and Leuthardt, F., Eur. J. Biochem. (1972) 31, 423-426).

Amino Acid Sequence↗

PubMed1991

The structure of the brain-specific rat aldolase C gene and its regional expression.

The rat aldolase C gene was isolated from a rat genomic DNA library. This gene comprises 9 exons and spans 3590 base pairs. A single copy of the gene occurs per haploid rat genome. The initiation of transcription occurs at two different sites. The cellular localization of aldolase C mRNA was determined in the central nervous system along with aldolase A mRNA by in situ hybridization. The result indicates the predominant expression of this gene in Purkinje cells of the cerebellar cortex, where aldolase A mRNA was rather repressed.

Amino Acid Sequence↗

PubMed1987

A new carbene based heterobifunctional reagent. Photochemical crosslinking of aldolase.

The synthesis of a new photoactivatable heterobifunctional crosslinking reagent, the N-oxysuccinimide ester of 2-carboxy-9-diazofluorene, is described. The ability of the parent chromophore 2-carbomethoxy-9-diazofluorene to insert into cyclohexane and methanol has been established. The reagent has been linked to aldolase and the stoichiometry determined. Photolysis of the probe-linked aldolase indicated that photolysis was very rapid and that the photolysed product was constituted of crosslinked dimer, trimer and tetramer. Increase in concentration of probe linked to aldolase followed by photolysis gave rise to largely tetramer and higher oligomers of aldolase. The use of this carbene-based reagent vis a vis arylazide-based reagent for studying protein crosslinking is discussed.

Cross-Linking Reagents↗

PubMed1991

The crystal structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster at 2.5 A resolution.

The structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster has been determined by X-ray diffraction at 2.5 A resolution. The insect enzyme crystallizes in space group P2(1)2(1)2(1) with lattice replacement with rabbit muscle aldolase as a search model has been employed to solve the structure. To improve the initial phases real space averaging, including phase extension from 4.0 to 2.5 A, has been applied. Refinement of the atomic positions by molecular dynamics resulted in a crystallographic R-factor of 0.214. The tertiary structure resembles in most parts that of the vertebrate aldolase from rabbit muscle. Significant differences were found in surface loops and the N- and C-terminal regions of the protein. Here we present the first aldolase structure where the functionally important C-terminal arm is described completely.

Animals↗

PubMed1993

Identification of an actin binding region in aldolase.

Fragmentation of the actin binding glycolytic enzyme, aldolase, with cyanogen bromide yields an 18K actin binding fragment which corresponds to residues 1-164 of the aldolase sequence. Within this fragment there is a region of sequence (residues 32-52) which is highly homologous to a region of sequence near the C-terminus of actin itself and which is also found in the actin binding domains of a number of other actin binding proteins. A synthetic peptide corresponding to the aldolase sequence 32-52 encompassing this region of homology binds to F-actin and specifically competes with native aldolase for binding to this cytoskeletal protein.

Actins↗

PubMed1985

Preliminary crystallographic investigation of rabbit liver aldolase.

Rabbit liver aldolase was purified by affinity elution on a CM52 ion exchanger. Crystals of rabbit liver aldolase suitable for X-ray diffraction experiments were grown from 45% saturated ammonium sulfate solution at 4 degrees C. The enzyme crystallizes in space group C222(1) having cell dimensions a = 377.02 A, b = 130.35 A, c = 80.04 A and diffracts to at least 3.5 A resolution. On the basis of a 55% solvent content there are eight aldolase tetramers in the unit cell. Rotational symmetry analysis to 6.7 A is consistent with the aldolase tetramers having a high degree of internal symmetry corresponding to point group 222. The crystallized enzyme is catalytically active.

Animals↗

PubMed1976

The age-dependent decrease in creatine kinase and aldolase activities in human striated muscle is not caused by an accumulation of faulty proteins.

In human striated muscle obtained in surgery, an age-dependent decrease in aldolase and creatine kinase specific activities and an increase in DNA content per wet weight was found. In the group of the elderly (64-84 years), the enzymes decreased by 40-60% when compared with a group between 24 and 47 years old, while DNA content rose by a factor of 1.53 indicating loss of tissue water. Titration of aldolase and creatine kinase molecules by specific antibodies against aldolase A and creatine kinase MM isozymes, respectively, revealed very little accumulation of aldolase cross-reacting materials in the old age group (1.13 fold), and no accumulation of inactive creatine kinase molecules. Similar conclusions can be drawn from thermostability analyses of these two enzymes. The data do not support the view that accumulation of modified proteins due to random errors or to post-translational alternations is a general or causative phenomenon of aging in human muscle tissue.

Adult↗