Properties of fructose 1,6-diphosphate aldolases from spores and vegetative cells of Bacillus cereus

Fructose 1,6-diphosphate aldolase from cells of Bacillus cereus appears to be typical Class II aldolase as judged by its functional and physical properties. Spore and vegetative cell aldolase had similar enzymatic, immunochemical, and heat resistance properties in the absence of calcium, but they differed in their thermal stabilities in the presence of calcium, their Stokes' radii, their mobility in acrylamide gel electrophoresis, and their molecular weights. The pH optimum for both enzymes was 8.5, and their K(m) with respect to substrate was 2 x 10(-3)m. Highly purified spore and vegetative cell aldolases were both heat labile with half-lives of 4 min at 53 C and pH 6.4. In the presence of 3 x 10(-2)m solution of calcium ions, the stability of the spore protein increased 12-fold but the vegetative form became more heat labile. The enhanced stability of the spore aldolase was not diminished by dialysis or gel filtration but was lost after chromatography on diethylaminoethyl cellulose at pH 7.4. Aldolase from vegetative cells exists in an equilibrium mixture of two molecular weights, 115,000 and 79,000 in the approximate ratio of 1:4, respectively. The molecular weight of spore aldolase is 44,000. Spore aldolase was more mobile during electrophoresis than its vegetative cell counterpart because of its smaller size.     

Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.     

Muscle aldolase decreases muscle FBPase sensitivity toward AMP inhibition

Muscle aldolase bound to muscle FBPase (K(d) = 8.7 microM) decreases the latter's sensitivity towards AMP inhibition. I(0.5) of muscle FBPase was increased from 0.06 microM to 0.65 microM when determined in the presence of 10 microM of muscle aldolase. In the presence of 10 microM of liver aldolase I(0.5) of liver FBPase was increased only twofold, from 11.0 microM to 21.7 microM. The effect of muscle aldolase on liver FBPase and liver aldolase on muscle FBPase is rather negligible. Aldolase slightly affected interaction of FBPase with magnesium ions decreasing K(a) and Hill constant (n). No effect of aldolase on FBPase pH optimum was observed.     

Purification and properties of the native form of rabbit liver aldolase. Evidence for proteolytic modification after tissue extraction.

Aldolase was purified from rabbit liver by affinity-elution chromatography. By taking precautions to avoid rupture of lysosomes during the isolation procedure, a stable form of liver aldolase was obtained. The stable form of the enzyme had a specific activity with respect to fructose 1,6-bisphosphate cleavage of 20-28 mumol/min per mg of protein and a fructose 1,6-bisphosphate cleavage of 20-28mumol/min per mg of protein and a frutose 1,6-bisphosphate/fructose 1-phosphate activity ratio of 4. It was distinguishable from rabbit muscle aldolase, as previously isolated, on the basis of its electrophoretic mobility and N-terminal analysis. Muscle and liver aldolases were immunologically distinct. The stable liver aldolase was degraded with a lysosomal extract to a form with catalytic properties resembling those reported for aldolase B4. It is postulated that liver aldolase prepared by previously described methods has been modified by proteolysis and does not constitute the native form of the enzyme.     

Two Class I Aldolases in the Green Alga Chara foetida (Charophyceae)

Aldolase activity of Chara foetida (Braun) could be separated into a minor (peak I) and a major peak (peak II) by ion-exchange chromatography on DEAE-cellulose. Affinity chromatography on P-cellulose resulted in highly purified aldolase preparations with specific activities of 3.2 and 4.8 units per milligram protein and molecular subunit masses of 37 and 35 kilodalton, as shown by SDS-PAGE, for the aldolase of peak I and peak II, respectively. Both aldolases belong to class I aldolase since the activity is not inhibited by 1 millimolar EDTA. The K_m (fructose-1,6-bisphosphate) values were 0.64 and 13.4 micromolar, respectively. The aldolase of peak I showed a 6.7 times stronger crossreaction with a specific antiserum against the cytosol aldolase of spinach than with an antiserum against the chloroplast aldolase of spinach. On the other hand the aldolase of peak II showed a 5.1 times stronger cross-reaction with the α-plastidaldolase antiserum than with the α-cytosol-aldolase antiserum. For algae this is the first separation of two class I aldolases. They are similar to the cytosol and chloroplast aldolases in higher plants, but different from a reported class I (Me²⁺ independent) and class II (Me²⁺ dependent) aldolase in other algae.     

Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.     

A selective reaction of fructose bisphosphate aldolase with fluorescein isothiocyanate in chicken muscle extracts

The present work describes the selective covalent modification of fructose bisphosphate aldolase in crude extracts of chicken breast muscle by fluorescein 5'-isothiocyanate (5'-FITC) at pH 7.0 and 35 degrees C. The modification was observed after 1 min while no other major soluble protein was labeled even after 30 min. We calculated that ca. one 5'-FITC molecule was incorporated into each aldolase tetramer after a 30 min reaction which resulted in a minimal loss of enzyme activity. The "native" structure of aldolase was required for the selective modification by 5'-FITC since high pH, high temperature, and ionic detergents either inhibited or prevented the reaction of 5'-FITC with aldolase. Certain metabolites (ATP, ADP, CTP, GTP, FBP) and erythrosin B also inhibited the 5'-FITC modification of aldolase. In contrast, F-6-P, AMP, NADH, and NAD(+) as well as free lysine and most importantly, the 6'-isomer of FITC exhibited no competition with 5'-FITC for the labeling of aldolase. Alone, the 6'-isomer of FITC did not exhibit preferential reaction when combined with aldolase. 5'-FITC-labeled and -unlabeled aldolases were not distinguished by their ability to bind to muscle myofibrils (MFs) or by their abilities to refold following reversible denaturation in urea. Structural analysis revealed that 5'-FITC-labeled a tryptic peptide corresponding to residues 112-134 in the primary structure of aldolase, a peptide that does not contain lysine, the amino acid believed to be the primary target of this reagent. Unlike chicken and rabbit muscle aldolases, chicken brain and liver aldolase isoforms along with several other aldolases derived from diverse biological sources did not exhibit this highly selective modification by 5'-FITC.     

Evidence for an interaction between fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase.

Three distinct lines of evidence suggest interaction and possible complex formation between fructose 1,6-biphosphate aldolase (EC 4.1.2.13) and fructose 1,6-biphosphatase (EC 3.1.3.11) from rabbit liver. (1) Fructose 1,6-biphosphatase, which does not contain tryptophan, causes changes in the fluorescence emission spectrum of tryptophan in rabbit liver aldolase. (2) Aldolase reduces the affinity of binding of Zn²⁺ to the two high-affinity sites of fructose 1,6-biphosphatase. (3) Gel penetration coefficients are decreased for both enzymes when they are tested together, as compared to the coefficients observed when each is tested separately. These interactions were not observed when either liver enzyme was replaced by the corresponding enzyme purified from rabbit muscle; this specificity for enzymes purified from the same tissue excludes effects attributable to the catalytic activities of the enzyme. Maximum interaction was observed in the pH range between 8.0 and 8.5 and appeared to require the presence of two fructose 1,6-biphosphatase tetramers per tetramer of aldolase. The change in fluorescence emission spectrum was also observed, to a smaller extent, when muscle fructose 1,6-biphosphatase was added to a solution of muscle aldolase.     

Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host

E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B.     

Quantitative expression studies of aldolase A, B and C genes in developing embryos and adult tissues of Xenopus laevis

We previously cloned cDNAs for all the members (A, B and C) of Xenopus aldolase gene family, and using in vitro transcribed RNAs as references, performed quantitative studies of the expression of three aldolase mRNAs in embryos and adult tissues. A Xenopus egg contains ca. 60 pg aldolase A mRNA and ca. 45 pg aldolase C mRNA, but contains only ca. 1.5 pg aldolase B mRNA. The percent composition of three aldolase mRNAs (A:B:C) changes from 56:1.5:42.5 (fertilized egg) to 54:10:36 (gastrula), to 71:14.5:14.5 (neurula) and to 73:20:7 (tadpole) during development. These results are compatible with the previous results of zymogram analysis that aldolases A and C are the major aldolases in early embryos, whose development proceeds depending on yolk as the only energy source. Aldolase B mRNA is expressed only late in development in tissues such as pronephros, liver rudiment and proctodeum which are necessary for the future dietary fructose metabolism, and the expression pattern is consistent to that in adult tissues. We also show that three aldolase genes are localized on different chromosomes as single copy genes.     

Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver.

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Extreme X-ray sensitive modification of type I aldolases by blue dye ligand chromatography

Aldolases purified by Blue dye ligand chromatography from a variety of vertebrate sources crystallize at room temperature in a habit similar to the monoclinic form of rabbit skeletal muscle aldolase. Crystals of aldolases thus purified including rabbit muscle aldolase are extremely sensitive to X-ray (Cu K alpha) radiation and shatter after short exposure to X-ray radiation (less than 5 min.). Crystals of aldolases purified by other techniques possess demonstrable diffraction patterns and are stable in the X-ray beam with lifetimes of the order of days. No clear distinction could be made on the basis of different biochemical assays between aldolases purified by Blue dye chromatography and those purified by other techniques.     

Hybridization between fructose diphosphate aldolase subunits derived from diverse biological systems: anomolous hybridization behavior of some aldolase subunit types

In the present studies we investigated the abilities of fructose diphosphate aldolase subunits derived from diverse biological sources to form stable heterotetramers with each other in vitro. Aldolase C subunits isolated from chicken brain readily "hybridized" with aldolase subunits derived from lobster muscle and wheat germ following reversible acid dissociation of mixtures of these enzymes; however, appreciable amounts of stable heterotetramers containing chicken C subunits and aldolase subunits isolated from two other invertebrates (Ascaris and squid) were not produced under the same conditions. In contrast to the situation with chicken C subunits, aldolase B subunits isolated from rat liver did not "hybridize" appreciably with lobster muscle or wheat germ aldolase subunits. The present observations are not consistent with the hypothesis that the abilities of different aldolase subunit types to form heterotetramers in vitro is governed solely by the evolutionary relationships which exist between the organisms from which the enzymes are derived.     

Tyrosine nitration impairs mammalian aldolase A activity

Protein tyrosine nitration increases in vivo as a result of oxidative stress and is elevated in numerous inflammatory-associated diseases. Mammalian fructose-1,6-bisphosphate aldolases are tyrosine nitrated in lung epithelial cells and liver, as well as in retina under different inflammatory conditions. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we now show that aldolase A is nitrated in human skin fibroblasts. To reveal the consequences of tyrosine nitration, we studied the impact of peroxynitrite on the glycolytic functions of aldolase A. A peroxynitrite concentration-dependent decrease in fructose-1,6-bisphosphate cleavage activity was observed with a concomitant increase in nitrotyrosine immunoreactivity. Both V(max) and the K(m) for fructose-1,6-bisphosphate decreased after incubation with peroxynitrite. Aldolase nitrotyrosine immunoreactivity diminished following carboxypeptidase Y digestion, demonstrating that tyrosine residues in the carboxyl-terminal region of aldolase are major targets of nitration. Aldolase A contains a carboxyl-terminal tyrosine residue, Tyr(363), that is critical for its catalytic activity. Indeed, tandem mass spectrometric analysis of trypsin-digested aldolase showed that Tyr(363) is the most susceptible to nitration, with a modification of Tyr(342) occurring only after nitration of Tyr(363). These tyrosine nitrations likely result in altered interactions between the carboxyl-terminal region and enzyme substrate or reaction intermediates causing the decline in activity. The results suggest that tyrosine nitration of aldolase A can contribute to an impaired cellular glycolytic activity.     

Site-directed mutagenesis of human aldolase isozymes: the role of Cys-72 and Cys-338 residues of aldolase A and of the carboxy-terminal Tyr residues of aldolases A and B

In order to elucidate the role of particular amino acid residues in the catalytic activity and conformational stability of human aldolases A and B [EC 4.1.2.13], the cDNAs encoding these isoenzyme were modified using oligonucleotide-directed, site-specific mutagenesis. The Cys-72 and/or Cys-338 of aldolase A were replaced by Ala and the COOH-terminal Tyr of aldolases A and B was replaced by Ser. The three mutant aldolases A thus prepared, A-C72A, A-C338A, and A-C72,338A, were indistinguishable from the wild-type enzyme with respect to general catalytic properties, while the replacement of Tyr-363 by Ser in aldolase A (A-Y363S) resulted in decreases of the Vmax of the fructose-1, 6-bisphosphate (FDP) cleavage reaction, activity ratio of FDP/fructose-1-phosphate (F1P), and the Km values for FDP and F1P. The wild-type and all the mutant aldolase A proteins exhibited similar thermal stabilities. In contrast, the mutant aldolase A proteins were more stable than the wild-type enzyme against tryptic and alpha-chymotryptic digestions. Based upon these results it is concluded that the strictly conserved Tyr-363 of human aldolase A is required for the catalytic function with FDP as the substrate, while neither Cys-72 nor Cys-338 directly takes part in the catalytic function although the two Cys residues may be involved in maintaining the correct spatial conformation of aldolase A. Replacement of Tyr-363 by Ser in human aldolase B lowered the Km value for FDP appreciably and also diminished the stability against elevated temperatures and tryptic digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of pig muscle aldolase. A comparative study

Aldolase with a specific activity of 10.8 units/mg protein was isolated from pig muscle. Its molecular weight was found to be 150,000. The optimum pH for the catalytic activity was 7.25 and the apparent temperature optimum was 313 K. The Km value was 2.9 X 10(-5) M with FDP as substrate, and 2.8 X 10(-3) M with F1P as substrate. The thermal stability of this pig muscle enzyme was higher than that of the rabbit muscle enzyme. The thermal inactivation of the pig aldolase did not show simple first-order kinetics. The higher conformational stability of the pig aldolase than that of the rabbit enzyme was demonstrated by its higher resistance to the denaturing effect of urea.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

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.     

Limited proteolysis of liver and muscle aldolases: Effects of subtilisin,cathepsin B,and Staphylococcus aureus protease

Limited proteolysis of rabbit liver and muscle aldolases by subtilisin and cathepsin B results in decreased catalytic activity, associated with the release of acid-soluble peptides from the COOH terminus. Analysis of the sequence of these peptides confirms the COOH-terminal sequence of the muscle enzyme and provides new information on the COOH-terminal sequence of the liver enzyme. As previously reported for muscle aldolase, cathepsin B releases mainly dipeptides from the COOH terminus of liver aldolase. The COOH-terminal sequence of rabbit liver aldolase is SerThrGlnSerLeuPheThrAla SerTyrThrTyr. The Gln-Ser bond is resistant to Staphylococcus aureus protease, which hydrolyzes a GluSer bond at the corresponding positions in the muscle enzyme.     

Two class II D-tagatose-bisphosphate aldolases from enteric bacteria

Escherichia coli, Salmonella enterica, Klebsiella pneumoniaeand Klebsiella oxytocawere found to contain two D-tagatose 1,6-bisphosphate (TagBP)-specific aldolases involved in catabolism of galactitol (genes gatY gatZ) and of N-acetyl-galactosamine and D-galactosamine (genes kbaY kbaZ,also called agaY agaZ). The two aldolases were closely related (> or = 53.8% identical amino acids) and could substitute for each other in vivo. The catalytic subunits GatY or KbaY alone were sufficient to show aldolase activity. Although substantially shorter than other aldolases (285 amino acids, instead of 358 and 349 amino acids), these subunits contained most or all of the residues that have been identified as essential in substrate/product recognition and catalysis for class II aldolases. In contrast to these, both aldolases required subunits GatZ or KbaZ (420 amino acids) for full activity and for good in vivo and in vitro stability. The Z subunits alone did not show any aldolase activity. Close relatives of these new TagBP aldolases were found in several gram-negative and gram-positive bacteria, e.g., Streptomyces coelicolor.