P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.     

Amino-acid Sequence Homology in the Muscle Aldolases from Sturgeons of Different Species

STUDIES on the primary structure of aldolases isolated from ox, pig and rabbit muscle show that the amino-acid sequence of fructose 1,6-diphosphate aldolase [EC 4.1.2.13] has been highly conserved throughout mammalian evolution¹. But comparison of the primary structure of the enzyme from these species with that from the muscle of a single North Sea sturgeon, presumably Acipenser sturio, indicated that although the proteins were homologous, a number of amino-acid replacements occurred between sturgeon aldolase and the aldolases of the phylogenetically distant mammalian species¹. As a knowledge of the nature and number of amino-acid replacements between homologous proteins caft provide information both about the functional role of individual residues and about evolution, further comparative studies of rabbit and sturgeon aldolases were undertaken and an account of the sequence homology around the active-site-lysine residue of aldolases from rabbit muscle, rabbit liver and the muscle of the river sturgeon of Eastern Canada, Acipenser fulvescens, has been given^2,3.     

Synthesis and cleavage of octulose bisphosphates with liver and muscle aldolases

Rabbit muscle aldolase was used to synthesize d-glycero-d-altro-octulose 1,8-bisphosphate and d-glycero-d-ido-octulose 1,8-bisphosphate. The products, isolated by ion-exchange chromatography, were characterized with the cysteine-sulfuric acid reaction and shown to be 90–95% pure by analysis for organic phosphorus and for dihydroxyacetone phosphate formed on cleavage with aldolase. The kinetic constants for synthesis and cleavage of these octulose bisphosphates with muscle and liver aldolases were determined. In the direction of cleavage both octulose bisphosphates were excellent substrates for liver aldolase, comparable to fructose 1,6-bisphosphate with respect to both V and K_m. With muscle aldolase the rate of cleavage was 1–5% of that with fructose bisphosphate and comparable to that with fructose 1-phosphate. In the direction of synthesis, ribose 5-phosphate was a better substrate than arabinose 5-phosphate for both the liver and muscle enzymes, although for both pentose phosphates the values of K_m fell in the range between 5 and 25 mm. It is concluded that reactions catalyzed by aldolase might account for the reported presence of these eight-carbon sugar phosphate esters in liver and in red cells.     

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.     

Fructose diphosphate aldolase-class I (Schiff base) fromMycobacterium tuberculosis H37Rv

An aldolase was partially purified from fermenter grownMycobacterium tuberculosis H₃₇Rv cells. The aldolase has a molecular weight of 150,000, possesses a tetrameric structure and cleaves both fructose diphosphate and fructose-1-phosphate, the former being cleaved 17 times faster. The enzyme was inactivated by treatment with NaBH₄ in the presence of fructose diphosphate or dihydroxyacetone, phosphate suggesting Schiff base formation during its catalytic function. Thiol reagents, EDTA and metal ions had no apparent effect on the aldolase activity. These results show that aldolase is of Class I type. However, this enzyme, unlike the mammalian Class I aldolase, was unaffected by carboxypeptidase A. N-ethylmaleiniide and dithionitrobenzoic acid.     

Properties of fructose 1,6-diphosphate aldolase from Drosophila melanogaster.

The amino acid composition and other properties of fructose 1,6-diphosphate aldolase from pupae of Drosophila melanogaster are reported and compared with those of other class I aldolases. Drosophila aldolase subunits contain only four residues of cysteine, five histidines, and two methionines. All four cysteine side chains react with 5,5′-dithiobis(2-nitrobenzoic acid) only in the presence of denaturating agent and are therefore thought to be buried within the molecule. With bromoacetate one carboxymethyl group is incorporated in the native enzyme with the loss of 90% of catalytic activity; inorganic phosphate is partially inhibiting this reaction. The near-uv absorption spectra of Drosophila and rabbit muscle aldolases are similar, the insect enzyme having higher absorbancies over the entire region corresponding to its higher tryptophan content. Circular dichroism-spectra of Drosophila aldolase indicate an α-helix content of 26%. Both the insect and vertebrate enzymes display marked tryptophan ellipticity bands between 290 and 300 nm.     

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.     

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.     

Preparation of isolated multiple forms of rabbit brain aldolase

1. A method is described for partial purification of the total aldolase of rabbit brain by ammonium sulphate precipitation followed by fractionation of the aldolase into five electrophoretically distinct forms. 2. The method allows a laboratory-scale preparation of the five aldolases. 3. With the exception of band 3 the fructose 1,6-diphosphate/fructose 1-phosphate activity ratios of the isolated multiple forms show a continuous increase from isoenzyme 1 to isoenzyme 5; this evidence supports the four-sub-unit model.     

Sites of cleavage of rabbit muscle aldolase by purified cathepsin M from rabbit liver

Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,6-bisphosphate but not with fructose 1-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH2-Ile-Ser-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOH-terminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme.     

Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes'' strain)

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca(2+), Zn(2+). It is a dimer with a molecular weight of approx. 70000, and the K(m) value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH(4) in the presence of substrate. The K(m) value for fructose diphosphate is about 20mum (although the Lineweaver-Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.     

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.     

Nucleo-cytoplasmic relationships of oestrogen receptors in rat liver during the oestrous cycle and in response to administered natural and synthetic oestrogen.

The mechanism of degradation of fructose-1,6-bisphosphate aldolase from rabbit muscle by the lysosomal proteinase cathepsin B was determined. Treatment of aldolase with cathepsin B destroys up to 90% of activity with fructose 1,6-bisphosphate as substrate, but activity with fructose 1-phosphate is slightly increased. Cathepsin L, another lysosomal thiol proteinase, and papain are also potent inactivators of aldolase, whereas inactivation is not caused by cathepsins D or H even at high concentrations, or by cathepsin B inhibited by leupeptin or iodoacetate. The cathepsin-B-treated aldolase shows no detectable change in subunit molecular weight, oligomer molecular weight or subunit interactions. Cathepsin B cleaves dipeptides from the C-terminus of th aldolase subunits. Four dipeptides are released sequentially: Ala-Tyr, Asn-His, Ile-Ser and Leu-Phe, and a maximum of five additional dipeptides may be released. There are indications that this peptidyldipeptidase activity of cathepsin B may be an important aspect of its action on protein substrates generally.     

TWO CLASS I ALDOLASES IN KLEBSORMIDIUM FLACCIDUM (CHAROPHYCEAE): AN EVOLUTIONARY LINK FROM CHLOROPHYTES TO HIGHER PLANTS1

Two fructose-bisphosphate aldolases(EC 4.1.2.13) from Klebsormidium flaccidum Silver, Mattox and Black-well were purified by affinity elution from phosphocellulose. The two enzymes were subsequently separated by HPLC on an anion-exchange column (QAE-silica). The aldolase eluting first represented 5% of the total activity; the other aldolase represented the remaining activity. The activity of the enzymes was not reduced by the presence of 1 mM EDTA or increased by 0.1 mM Zn²⁺, establishing their character as class I type (Me²⁺ independent) aldolases. The K_m(fructose-1,6-bisphosphate) values were 1.7 and 34.7 μM for the enzyme eluting first and second, respectively, from the QAE-silica column. The subunit molecular masses, as determined by SDS-PACE, were 40.5 and 37 kD; the specific activities of the purified enzymes were 7.9 and 24.7 · mg^?1 protein, respectively. The two aldolases of K. flaccidum are homologous to the cytosol and chloroplast specific isoenzymes of higher plants by several criteria and are therefore probably located in the same cellular compartments in K. flaccidum. The K_m and specific activity for the chloroplast aldolase of K. flaccidum are three times higher than for the chloroplast aldolase of higher plants, a remarkable difference. Immunotitration with specific antisera against the chloroplast aldolase of Chlamydomonas reinhardtii Dangeard and spinach showed that the chloroplast aldolase of K. flaccidum was immunochemically intermediate in structure to the respective aldolases of C. reinhardtii and higher plants. K. flaccidum is the second species of Charophyceae (besides Chara foetida Braun) with two class I aldolases as in higher plants whereas two species of Chlorophyceae have only one class I aldolase and, under some conditions, an additional class II (Me²⁺ dependent) aldolase. Thus, aldolases may turn out, in addition to the known enzymes of glycolate conversion and urea degradation, be a novel enzyme system to evaluate algal evolution along with cytological features.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

A comparative study of aldolase from human muscle and liver

Aldolase was purified from human skeletal muscle and human liver by techniques capable of processing large quantities (10-20kg) of tissue. The methods used also proved convenient for isolating aldolase on a large scale from other mammalian and avian sources. Aldolase from both human liver and muscle was crystallized; each gave two crystalline forms, depending on the conditions of crystallization. X-ray studies on the muscle aldolase crystals suggest a close structural similarity between human and rabbit muscle aldolase. Aldolases from human muscle and liver have similar pH optima and pH stability but their stability to heat treatment differs. The effect of heat on the enzymes may therefore provide an easy means of distinguishing them. The kinetic constants K(m) and k(cat.) for these aldolases are similar to other mammalian aldolases. Amino acid analyses and tryptic peptide ;mapping' show that the primary structures of the two aldolases differ greatly.     

Aldolases of the lactic acid bacteria

Reciprocal qualitative and quantitative immunological experiments employing an anti-Pediococcus cerevisiae aldolase serum confirmed many of the interspecific relationships demonstrated previously among lactic acid bacteria with antisera prepared against the Streptococcus faecalis fructose diphosphate aldolase. The extent of immunological relatedness observed between the Lactobacillus and Pediococcus aldolases was markedly greater than that noted between Pediococcus and Streptococcus aldolases indicating that the pediococci share closer phylogenetic ties with the rod-shaped lactobacilli than with their spherical counterparts in the streptococci. In addition to confirming the existence of definitive, but distant, relationships between the lactic acid bacteria and certain gram positive nonsporeforming anaerobes, immunological cross-reactivity was also demonstrated between the pediococcal aldolases and those of Aerococcus viridans.This paper is dedicated with deepest appreciation to Prof. Roger Y. Stanier on the occasion of his 60th birthday in token of what his friendship and guidance have meant to me. — J. L.     

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.     

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.     

Fructose diphosphate aldolase from Mycobacterium smegmatis. Purification and properties.

Fructose diphosphate aldolase has been purified to homogeneity from Mycobacterium smegmatis. Physicochemical studies showed that the enzyme is a tetramer of molecular weight 158,000. Mycobacterium smegmatis aldolase, though a bacterial enzyme, possesses properties similar to other class I aldolases. Inactivation of the enzyme by sodium borohydride in presence of dihydroxyacetone phosphate suggested the formation of a Schiff-base intermediate.