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PubMed1983

Physicochemical characterization of a fast refolding monomeric class I fructose-1,6-bisphosphate aldolase from Staphylococcus aureus.

The class I fructose-1,6-bisphosphate aldolase from Staphylococcus aureus is proposed as a good candidate for thermodynamic and kinetic studies on protein folding. The monomeric enzyme (molecular weight 35 000 +/- 1000) has been previously described as 'unusually heat-stable' [F. Götz et al. (1980) Eur. J. Biochem. 108, 295-301]. In the present paper we show that the enzyme is reversibly denatured at relatively low temperature (26-39 degrees C), as determined by protein fluorescence and far ultraviolet circular dichroism; the van't Hoff enthalpy of the thermal unfolding is 355 +/- 63 kJ/mol. The dichroic absorption shows that the aldolase is extensively unfolded in 6 M guanidine/HCl. Complete reactivation of the guanidine-denatured enzyme in the test solution is extremely fast (less than 10 s in the temperature range from 24.6 degrees C to 7.7 degrees C). Reactivation ought to be much slower if isomerization reactions around at least some of the ten Xaa-Pro peptide bonds were rate-limiting for reactivation.

Animals↗
PubMed1988

Semisynthetic derivatives of inositol 1,4,5-trisphosphate substituted at the 1-phosphate group. Effects on calcium release from permeabilized guinea-pig parotid acinar cells and comparison with binding to aldolase A.

Derivatives of inositol 1,4,5-(tris)phosphate [Ins(1,4,5)P3] substituted at phosphate 1 were compared with respect to their calcium releasing effect in permeabilized guinea pig parotid acinar cells and to their inhibitory action on aldolase A. sn-Glycero(3)-1-phospho-D-myo-inositol-4,5-(bis)phosphate, but also glycolaldehyde(2)-1-phospho-D-myo-inositol-4,5-(bis)phosphate [GcaPIns(4,5)P2] and its derivative N-octyl-aminoethanol(1)-1-phospho-D-myo-inositol-4,5-(bis)phosphate stimulated calcium release and inhibited aldolase A. The relative efficacy of the different derivatives of Ins(1,4,5)P3 was similar for both effects. N-Hydroxyethyl-2-aminoethanol(1)-1-phospho-D-myo-inositol-4,5-(bis)phosp hate [HeAetPIns(4,5)P2], another derivative of GcaPIns(4,5)P2 was considerably less effective on both parameters than the other Ins(1,4,5)P3 derivatives. Although the concentration leading to half-maximal activation of calcium release varied from 1.7 microM for Ins(1,4,5)P3 to 128 microM for HeAetPIns(4,5)P2, the maximal effect was the same for all derivatives. The results indicate that the 1-phosphate group of Ins(1,4,5)P3 can be modified without or with only minor loss of biological activity. This may be utilized for future studies aiming at elucidating the putative Ins(1,4,5)P3 binding site.

Animals↗
PubMed1993

Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site.

Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527-2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse-phase HPLC. Mass spectroscopy, 1H- and 13C-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of the two lysine residues: [formula: see text]

Amino Acids↗
PubMedDate not supplied

Decrease of aldolase and pyruvate kinase activity in erythrocytes of individuals with male hypogonadism as an expression of lack of androgen influence on the bone marrow.

The authors have demonstrated the statistically significant decrease of aldolase and pyruvate kinase activity in erythrocytes in a group of men with hypogonadism and with a significant defect of gonad endocrinologic function. A similar phenomenon appeared in erythrocytes in a group of sexually mature rabbits after castration. In the course of substitutional treatment with testosterone esters given intramuscularly, the increase of activity characteristic for both examined glycolytic enzymes was observed in both experimental groups, i.e. the patients and the animals. The pyruvate kinase increase, however, was not statistically significant in the examined group of men, which resulted from the small size of the group. The authors suggest the existence of testosterone influence on regular activity of bone marrow glycolytic enzymes, and consequently on the mature erythrocytes. The decrease of aldolase and pyruvate kinase activity in erythrocytes of patients with male hypogonadism may decrease the erythrocytes valence. This ist another biochemical argument for the need of conducting long-term substitutional therapy in cases of male hypogonadism.

Adult↗
PubMed1984

Developmental changes in lactate dehydrogenase and aldolase activity of the A2G-adr mouse with abnormal muscle function: further comparison with the 129Re-dy mutant.

Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G-adr and 129Re-dy, with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G-adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re-dy muscle confirm the findings of other workers.

Aging↗
PubMed1996

Studies with type I aldolase to understand fructose intolerance and combat parasitic disease.

A structural study of the type I aldolases has been carried out to examine the isozyme specificity of these enzymes and the potential for designing specific inhibitors. Natural mutations in these aldolase enzymes are associated with haemolytic anaemia and fructose intolerance. It has also been proposed that inhibition of the parasitic version of the enzyme may provide a new lead in the design of drugs against malaria and sleeping sickness. X-ray crystallographic data is used with molecular modelling techniques to investigate the structural properties of these enzymes.

Animals↗
PubMed1997

Immune versus natural selection: antibody aldolases with enzymic rates but broader scope.

Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.

Animals↗
PubMed1988

Aldolase activity of a Plasmodium falciparum protein with protective properties.

Immunization with a 41-kilodalton blood stage antigen (p41) of Plasmodium falciparum induces immunity to malaria in monkeys. However, antigenic polymorphism and repetitive amino acids commonly found in protective antigens complicate vaccine development. The gene encoding p41 has now been cloned and analyzed. Sequencing and hybridization studies revealed that the gene structure is highly conserved in 14 parasite isolates from three continents. This finding and the lack of repetitive amino acids in the translated DNA sequence may indicate that p41 has an essential function. In this study the protein was found to be 60 percent homologous to the key glycolytic enzyme aldolase from vertebrates, and the affinity-purified p41 protein from parasites showed aldolase activity.

Animals↗
PubMed2006

Effects of depleting the essential central metabolic enzyme fructose-1,6-bisphosphate aldolase on the growth and viability of Candida albicans: implications for antifungal drug target discovery.

The central metabolic enzyme fructose-1,6-bisphosphate aldolase (Fba1p) catalyzes a reversible reaction required for both glycolysis and gluconeogenesis. Fba1p is a potential antifungal target because it is essential in yeast and because fungal and human aldolases differ significantly. To test the validity of Fba1p as an antifungal target, we have examined the effects of depleting this enzyme in the major fungal pathogen Candida albicans. Using a methionine/cysteine-conditional mutant (MET3-FBA1/fba1), we have shown that Fba1p is required for the growth of C. albicans. However, Fba1p must be depleted to below 5% of wild-type levels before growth is blocked. Furthermore, Fba1p depletion exerts static rather than cidal effects upon C. albicans. Fba1p is a relatively abundant and stable protein in C. albicans, and hence, Fba1p levels decay relatively slowly following MET3-FBA1 shutoff. Taken together, our observations can account for our observation that the virulence of MET3-FBA1/fba1 cells is only partially attenuated in the mouse model of systemic candidiasis. We conclude that an antifungal drug directed against Fba1p would have to be potent to be effective.

Animals↗
PubMed1971

Properties of aldolase from Francisella tularensis.

The aldolase of Francisella tularensis resembles Class II aldolases in its requirement for divalent ions and its inactivation by metal chelating agents. Cysteine and other reducing agents stimulated the activity of the enzyme.

Chelating Agents↗
PubMed1985

Fructose 1,6-bisphosphate aldolase activity is essential for synthesis of alginate from glucose by Pseudomonas aeruginosa.

We have isolated a mutant of Pseudomonas aeruginosa deficient in fructose 1,6-bisphosphate aldolase activity. This mutant, similar to the mutants deficient in any of the Entner-Doudoroff pathway enzymes, does not allow appreciable alginate formation from glucose and gluconate, but allows alginate synthesis from mannitol and fructose. This suggests that glucose and gluconate must be converted to fructose 1,6-bisphosphate via the Entner-Doudoroff pathway enzymes and fructose 1,6-bisphosphate aldolase.

Alginates↗
PubMed1991

The Escherichia coli ts8 mutation is an allele of fda, the gene encoding fructose-1,6-diphosphate aldolase.

The ts8 mutant of Escherichia coli has previously been shown to preferentially inhibit stable RNA synthesis when shifted to the nonpermissive temperature. We demonstrate in this report that the ts8 mutation is an allele of fda, the gene that encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase. We show that ts8 and a second fda mutation, h8, isolated and characterized by A. Böck and F. C. Neidhardt, are dominant mutations and that they encode a thermolabile aldolase activity.

Alleles↗
PubMed1993

Cloning, sequencing, and characterization of the gene encoding the class I fructose-1,6-bisphosphate aldolase of Staphylococcus carnosus.

fda from Staphylococcus carnosus TM300, encoding the class I fructose-1,6-bisphosphate aldolase, was cloned in Escherichia coli and sequenced. The 888-nucleotide open reading frame encoding a protein with an M(r) of 32,855 had an E. coli-like promoter sequence. Plasmids containing fda complemented E. coli NP315 (Fda-). Expression of fda in S. carnosus led to a six- to eightfold increase in aldolase production and activity; low levels of glucose in the growth medium stimulated activity.

Amino Acid Sequence↗
PubMed2006

Binding of AlF-C, an Orc1-binding transcriptional regulator, enhances replicator activity of the rat aldolase B origin.

A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.

Amino Acid Sequence↗
PubMed1993

The MEF-3 motif is required for MEF-2-mediated skeletal muscle-specific induction of the rat aldolase A gene.

The rat aldolase A gene contains two alternative promoters and two alternative first exons. The distal promoter M is expressed at a high level only in skeletal muscle. Previous in vitro transfection studies identified the region from -202 to -85 as an enhancer that is responsible for dramatic activation during the differentiation of chicken primary myoblasts. This enhancer contains an A/T-rich sequence resembling the MEF-2 motif, which is an important element of muscle enhancers and promoters. In this study, we demonstrate that the MEF-2 sequence is essential but not sufficient for the activity of the enhancer. Another region required for the activity was recognized by a nuclear factor, tentatively named MAF1. MAF1 was found in both muscle cells and nonmuscle cells, and MAF1 from both cell types was indistinguishable by gel retardation and DNase I footprint experiments. The sequence required for MAF1 binding is very similar to the MEF-3 motif, which is an element of the skeletal muscle-specific enhancer of the cardiac troponin C gene. Because MAF1 and MEF-3 are closely related in both recognition sequence and distribution, MAF1 and MEF-3 probably represent the same nuclear factor which may play an important role in muscle gene transcription. Thus, the muscle-specific induction of the aldolase A gene is governed by muscle-specific MEF-2 and existing MEF-3 (MAF1).

Animals↗
PubMed1977

Comparison of aldolase isozymes in placenta, HeLa cells, and human fibroblast cultures.

The aldolase specific activity of the human carcinoma cell line, HeLa, against fructose 1,6-diphosphate as substrate is 4- to 5-fold greater than the specific activity of diploid human fibroblast cultures derived from skin and lung. HeLa aldolase is isozyme is predominantly the A type and its substrate preferences resemble human placenta. These findings provide further support for the oncofetal enzyme consitution of HeLa cells.

Cells, Cultured↗
PubMed1980

Aldolase isoenzyme patterns during human ontogeny and in lung, kidney and breast cancer.

Enzyme patterns characteristic of fetal tissue have been noted in some experimental tumor models, particularly in hepatomas. In this study we undertook to determine whether biochemical evidence of a similar reversion could be detected in tumors of other human organs. As marker, we chose to use the aldolase isoenzymes A, B and C, for which distinct adult and fetal tissue patterns have been described. Using monospecific antibodies, we determined the aldolase isoenzyme pattern in a variety of human organs ranging in age from 14 to 40 weeks of gestation, in the 2- to 3-month postnatal period and in adults. In addition, 19 breast cancers, 19 primary lung cancers and 8 kidney cancers were examined. Our studies on breast cancer revealed three apparently distinct groups -- one showing primarily the A isoenzyme type (6 cases), a second containing mainly A with considerable quantities of B and C isoenzymes (9 cases) and a third group (4 cases) which may contain a different isoenzyme altogether since the combined activity of the three known forms was less than 100% in each case. In lung cancer, fetal characteristics could be substantiated since in fetal and adult lung tissue, the isoenzyme pattern is almost identical; 3 out of 19 cases showed substantial quantities of the B isoenzyme. In kidney tumors, a reversion to the A form with an appreciable fraction of the C form was found, which is similar to the fetal pattern.

Adenocarcinoma↗
PubMed1978

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH.

Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.

Alkalies↗