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PubMed1986

The complete amino Acid sequence for the anaerobically induced aldolase from maize derived from cDNA clones.

A cDNA library was synthesized from maize anaerobic root mRNA and screened with cDNA specific to the anaerobically induced Zea mays cytoplasmic aldolase. At least 1% of the cDNA of the library corresponded to maize cytoplasmic aldolase. The sequence of four overlapping cDNA clones encoded a protein of molecular weight 38,611 homologous to aldolase. These cDNAs were polymorphic at three bases and one of these cDNAs had a different, shorter 3'-untranslated region. No known eukaryotic poly(A) addition site was detected. The derived amino acid sequences of maize was compared to the sequence of aldolase of trypanosome, Drosophila, and two mammalian isozymes, A and B. Of these, maize cytoplasmic aldolase was found to have the highest homology (55%) with rabbit aldolase A.

Journal Article↗
PubMed1998

Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg-1 was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.

Amino Acid Sequence↗
PubMed1993

A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities.

A protein encoded by sulD, one of four genes in a previously cloned folate biosynthetic operon of Streptococcus pneumoniae, had been shown to harbor 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity. This SulD protein was purified and shown now to harbor also dihydroneopterin aldolase activity. The bifunctional protein therefore catalyzes two successive steps in folate biosynthesis. The aldolase activity can be ascribed to the N-terminal domain of the SulD polypeptide, and the pyrophosphokinase activity can be ascribed to the C-terminal domain. Homologs of the dihydroneopterin aldolase domain were identified in other species, in one of which the domain was encoded as a separate polypeptide. The native SulD protein is a trimer or tetramer of a 31-kDa subunit, and it dissociated reversibly after purification. Dihydroneopterin aldolase activity required the multimeric protein, whereas pyrophosphokinase was expressed by the monomeric form. With purified SulD, the amount of 6-hydroxymethyl-7,8-dihydropterin product formed by the aldolase was proportional to the fourth power of the enzyme concentration, as expected for a reversibly dissociating tetramer. By identifying the gene encoding dihydroneopterin aldolase, this work extends our understanding of the molecular basis of the folate biosynthetic system common to many organisms.

Aldehyde-Lyases↗
PubMed1966

Isolation of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase Activity.

Böck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Isolation of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase activity. J. Bacteriol. 92:464-469. 1966.-A mutant of Escherichia coli was isolated which was able to grow in rich medium at 30 C but not at 40 C. Upon exposure to 40 C, the cells immediately stopped ribonucleic acid (RNA) and deoxyribonucleic acid synthesis, but protein synthesis continued at a diminished rate for a short time. Addition of chloramphenicol did not release RNA synthesis from inhibition at 40 C. Synthesis of beta-galactosidase could be induced at high temperature despite the presence of glucose in the medium, indicating a lesion in glucose catabolism. Of many catabolic enzymes tested in cell-free extracts, only fructose-1,6-diphosphate aldolase activity appeared to be altered in the mutant cells. No activity was demonstrable in extracts of mutant cells grown at either 30 or 40 C, but determination of glucose-oxidation patterns revealed that the enzyme is probably active in vivo at 30 C. Temperature-resistant secondary mutants were found to have partially or fully restored aldolase activity, and temperature-resistant recombinants had normal aldolase activity, indicating that the growth pattern and the altered aldolase had a common genetic basis. Linkage data permitted the assignment of an approximate map location for the mutated aldolase gene.

Journal Article↗
PubMed1986

Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase.

Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.

Amino Acid Sequence↗
PubMed1988

The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase.

The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.

Amino Acid Sequence↗
PubMed1996

Rapamycin inhibits aldolase A expression during human lymphocyte activation.

Rapamycin (RAPA) strongly inhibits lymphocyte activation and proliferation, but does not affect most of the activation-related gene expression at the mRNA level. In order to understand the mechanism of action of RAPA and to gain further insights in lymphocyte signalling which is impaired by RAPA, we screened for RAPA-sensitive genes using differential hybridization. The expression of human aldolase A gene was found to be inducible during T and B cell activation, and the induction was repressed by RAPA at both the mRNA and enzymatic levels. The other two important immunosuppressants, cyclosporin A and FK506, also inhibited the mitogen-induced upregulation. However, none of these three drugs inhibited the constitutive expression. There was no fluctuation of aldolase A expression during the cell cycle, and RAPA failed to block the first cell cycle after synchronization in Jurkat cells. However, the second cycle was hampered by RAPA, and this was correlated with the inhibition of aldolase A expression during this later stage. Since aldolase A is a key enzyme in glycolysis and lymphocytes mainly depend on glycolysis for energy supply, the data from this study suggest that aldolase A might be one of the downstream targets of RAPA. The inhibition of the enzyme upregulation might deprive the cells of additional supply of energy, and prevent the cells from entering an optimal status for proliferation.

Cyclosporine↗
PubMed2000

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.

Animals↗
PubMed1991

Immunochemical and immunohistochemical studies on three aldolase isozymes in human lung cancer.

The aldolase isozymes A, B, and C in tumor tissues (63) and sera (104) of patients with lung cancer were determined with an enzyme immunoassay system, compared with normal lung tissues (13), and the sera of normal healthy subjects (100). Tissue aldolase A and C concentrations were enhanced in 83% (52/63) and 51% (32/63) of patients with lung cancer, respectively, regardless of histologic type or stage (P less than 0.01). But aldolase B was not elevated in tissue levels. In the sera of patients with lung cancer, there were no significant elevations of the isozymes. Immunohistochemically aldolase A and C stained more intensely in the cytoplasm of lung cancer cells than those in normal tissues. These results indicate lung cancer cells contain enhanced tissue levels of aldolase A and C.

Adolescent↗
PubMed2007

Relationship of complex spike synchrony bands and climbing fiber projection determined by reference to aldolase C compartments in crus IIa of the rat cerebellar cortex.

Synchronous complex spike (CS) activity occurs most often among cerebellar Purkinje cells located in a narrow longitudinal (parasagittal) strip of cortex (synchrony band). The relationship of the anatomical organization of the olivocerebellar projection to these synchrony bands has not been investigated in detail. Thus, we studied this relationship by using the aldolase C (zebrin II) expression pattern, another landmark for the cerebellar longitudinal organization, as a reference frame in rat crus IIa. Crus IIa consists of 10 aldolase C-positive and -negative longitudinal compartments. Aldolase C labeling after multiple-electrode recording of CSs indicated that in lateral crus IIa (compartments 5+ to 7+) synchrony bands were generally constrained to single compartments. In contrast, in medial crus IIa (compartments 4a- to 5a-) the synchrony within and across the compartments was much higher than in lateral crus IIa, resulting in wide synchrony bands covering multiple compartments. Retrograde labeling of olivary neurons by injections of biotinylated dextran amine into aldolase C compartments in crus IIa showed that compartments in medial crus IIa were all innervated by the caudal part of the medial accessory olive. On the other hand, each aldolase C compartment in the lateral crus IIa was innervated by a region in a different subnucleus in the rostral inferior olive. These regions in different subnuclei were located close to each other. These results suggest that CS synchrony bands reflect the olivocerebellar compartmental projection pattern and neuronal coupling within a particular olivary subnucleus, and that medial and lateral crus IIa may be functionally distinct.

Action Potentials↗
PubMed2006

Identification of an anti-aldolase autoantibody as a diagnostic marker for diabetic retinopathy by immunoproteomic analysis.

Circulating autoantibodies specific for retinal proteins are associated with retinal destruction in patients with diabetic retinopathy (DR). In this study, we screened diabetic sera for the presence of anti-retinal autoantibodies with an aim of developing diagnostic markers for DR. Immunoblot analysis of DR patients' sera with human retinal cytosolic proteins revealed a higher incidence of anti-retinal autoantibodies, compared to normal blood donors or diabetic patients without DR. Anti-retinal protein autoantibody profiles of DR patient sera were obtained by 2-DE immunoblot analysis. Specifically, 20 protein spots reactive with DR patient sera were identified by ESI-MS/MS. Of these spots, 14 were specific for DR patients, and 4 reacted with both non-proliferative DR (non-PDR) and PDR sera. The anti-aldolase autoantibody was selected as a DR marker candidate, and specific reactivity of DR patient sera was confirmed by immunoblot analysis with rabbit aldolase. The serum anti-aldolase autoantibody level was measured by ELISA. DR patients showed significantly higher autoantibody levels than normal donors or diabetic patients without retinopathy. However, no significant differences were observed between non-PDR and PDR patients, suggesting that the level of anti-aldolase autoantibody is not determined by the severity of retinopathy in diabetic patients. Our data collectively demonstrate that the anti-aldolase autoantibody serves as a useful marker for DR diagnosis.

Adult↗
PubMed1993

Aldolase-tubulin interactions: removal of tubulin C-terminals impairs interactions.

Aldolase copelleted with taxol stabilized microtubules with a Bmax = 0.74 moles of aldolase per mole of tubulin dimer. Removal of the carboxy terminals from microtubules with limited subtilisin digestion, decreased binding to 0.16 moles of aldolase per mole of tubulin dimer. Aldolase inhibited subtilisin cleavage of the C-terminals while triose phosphate isomerase, an enzyme that does not interact with microtubules, did not affect subtilisin activity. These data indicate that the carboxy terminals are involved in tubulin-aldolase interactions.

Animals↗
PubMed2000

Structures of type 2 peroxisomal targeting signals in two trypanosomatid aldolases.

Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These "PTS2 dimers", which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.

Amino Acid Sequence↗
PubMed1976

Differentiation of epithelial cells in human jejunum: localization and quantification of aminopeptidase, alkaline phosphatase and aldolase isozymes in tissue sections.

Sections from human jejunum were stained histochemically for aminopeptidase and alkaline phosphatase and the aldolase isozymes were detected with the mixed aggregation immuno-cytochemical technique. All enzyme concentrations increased from the bottom to the upper part of the crypt. The concentration of aldolase-A per cell was the same in the upper part of the crypt and the villus, whereas the concentration of the other three enzymes was still higher. Therefore, high amounts of aldolase-B, aminopeptidase and alkaline phosphatase are present in cells highly active in absorption in a fashion similar to that found in the proximal tubule cells of kidney. The relatively undifferentiated cells of the crypts contained both aldolase-A and aldolase-B. Alkaline phosphatase gains its full activity later than aminopeptidase. The synthesis of microvillar membrane enzymes comes to an end earlier than that of the cytosol enzymes.

Alkaline Phosphatase↗
PubMed1975

The cellular distribution of aldolase isozymes in rat kidney and brain determined in tissue sections by the immuno-histochemical method.

The use of the immuno-histochemical method permits the localization of aldolase isozymes in tissue sections. Upon incubating a section with a monomer-specific antiserum, isozymes containing that monomer remain in the section, whereas other cytoplasmic enzymes diffuse out of the section. If soluble antigen is added subsequently, it is bound by the tissue-bound antibody. These antibody fixed aldolases can then be stained by the use of a tetrazolium test linked to substrate hydrolysis. In this way it was demonstrated that isozymes of aldolase containing mostly the A monomer are predominantly localized in the distal tubules, the collecting tubules, the vessels and capillaries of the kidney, the ganglia, the Purkinje cells, the neurons, the white matter and the chorioid plexus of the brain. Aldolase containing mostly B-monomers were found in the proximal tubules. Aldolase isozymes particularly rich in C-monomers were seen in the nervus opticus, the pia mater, the vessels of cerebrum and the molecular layer of the cortex cerebelli.

Brain↗
PubMed1983

Similarities in properties, content, and relative rates of synthesis of fructose-P2 aldolase in livers of fed and starved rats.

The present work gives evidence that, in contrast to the situation reported by Pontremoli et al. for the rabbit (Proc, Natl. Acad. Sci. U.S.A. 76, 6323-6325, 1979; Arch. Biochem. Biophys. 203, 390-394, 1980; Proc. Natl. Acad. Sci. U.S.A, 79, 5194-5196, 1982), starvation for as long as 3 days does not cause intracellular covalent modification and inactivation of fructose-P2 aldolase molecules in rat liver cells. This conclusion is based on our observations that liver aldolase molecules isolated from fed and starved rats in the presence of proteolytic inhibitors were not distinguished on the basis of specific catalytic activity, electrophoretic mobility, subunit molecular weight, NH2-terminal structure, or COOH-terminal structure. Further, the approximate 40% loss in rat liver mass which occurred during the 3-day fast was not associated with appreciable changes in the content of aldolase and most other abundant cytosolic proteins per gram of rat liver, as judged by electrophoretic analysis of 100 000-g soluble fractions of liver extracts. Finally, a 3-day fast had no appreciable effect on the relative rates of synthesis of aldolase and most other abundant cytosolic proteins in rat liver. Our findings suggest that nutrient deprivation has no preferential effect on the concentration or metabolism of aldolase in rat liver cells.

Animals↗
PubMed1988

Archaebacterial class I and class II aldolases from extreme halophiles.

Both, class I (Schiff-base forming) and class II (metal requiring) fructose biphosphate aldolases were found to be distributed among halophilic archaebacteria. The aldolase activity from Halobacteriium halobium, H. salinarium, H. cutirubrum, H. mediterranei and H. volcanii exhibited properties of a bacterial class II aldolase as it was metal-dependent for activity and therefore inhibited by EDTA. In contrast, aldolase from H. saccharovorum, Halobacterium R-113, H. vallismortis and Halobacterium CH-1 formed a Schiff-base intermediate with the substrate and therefore resembled to eukaryotic class I type. The type of aldolase did not vary by changes in the growth medium.

Dihydroxyacetone Phosphate↗
PubMed1997

An estimate of divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata by aldolase and triose phosphate isomerase clocks.

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and sequenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

Amino Acid Sequence↗