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PubMed1992

Intracellular and serum levels of aldolase activity in B chronic lymphocytic leukemia.

Intracellular and serum activities of aldolase (ALS) were biochemically determined in lymphocyte subpopulations from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL). Aldolase activity was significantly lower in T cells of CLL than in normal T cells (2.9 +/- 1.5 vs. 4.7 +/- 2.1 Sigma Units (SU)/6 x 10(6) cells, p < 0.05). The aldolase activity also was significantly (p < 0.001) lower (3.1 +/- 1.9 SU/6 x 10(6) cells) in CLL B lymphocytes than in normal B lymphocytes (18.1 +/- 6.5 SU/6 x 10(6) cells). Moreover, the serum levels of ALS in all patients with B-CLL were higher than that in normal subjects (8.1 +/- 5.8 vs. 2.2 +/- 0.8 SU/ml, p < 0.02). Our findings demonstrate that T lymphocytes from patients with B-CLL display enzyme activity different from that of normal T cells. This may reflect the abnormal maturity of the residual T cell population in B-CLL.

Adult↗
PubMed1991

Identification of a splice-site mutation in the aldolase B gene from an individual with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients exhibit a deficiency of fructose 1-phosphate aldolase (aldolase B), the isozyme expressed in tissues that metabolize fructose. The eight protein-coding exons, including splicing signals, of the aldolase B gene from one HFI patient were amplified by PCR. Dot-blot hybridization of the amplified DNA with allele-specific oligonucleotide (ASO) probes revealed a previously described A149P mutation in one allele from the proband. The mutation in the other allele was identified by direct sequencing of the double-stranded PCR-amplified material from the proband. The nucleotide sequence of exon 9 revealed a 7-base deletion/1-base insertion (delta 7 + 1) at the 3' splice site of intron 8 in one allele. This mutation was confirmed by cloning PCR-amplified exon 9 of the proband and determining the sequence of each allele separately. ASO analysis of 18 family members confirmed the Mendelian inheritance of both mutant alleles. The implications of this unique splice-site mutation in HFI are discussed.

Adolescent↗
PubMed1990

Hereditary fructose intolerance caused by a nonsense mutation of the aldolase B gene.

The nucleotide sequence of a patient's aldolase B gene was determined and showed a substitution of a single nucleotide (C----A) at position 720 in the coding region, which resulted in the 240th amino acid, a cysteine, being changed to a stop codon (TGC----TGA). By an allele-specific oligonucleotide probe and polymerase chain reaction, the patient was shown to be homozygous for the mutation. To examine whether this mutation causes functional defect of the enzyme, the activity of the aldolase B from the patient, expressed in Escherichia coli by using expression plasmid, was measured. No activity was observed, and the predicted product was recovered from E. coli expression plasmid, indicating that this nonsense mutation was the cause of aldolase B deficiency.

Base Sequence↗
PubMed1990

Construction and properties of active chimeric enzymes between human aldolases A and B. Analysis of molecular regions which determine isozyme-specific functions.

To study the structure/function relationships of human aldolase isozymes, particularly isozyme-specific functions, we constructed Escherichia coli expression plasmids for six BA chimeric enzymes (BA34, BA108, BA137, BA212, BA306, and BA306*), each composed of the N-terminal side of isozyme B and the C-terminal side of isozyme A, and one BAB chimeric enzyme which contains a fragment of isozyme A (residues 213-306) inserted in between the N-terminal and the C-terminal fragments of isozyme B. They were transfected into E. coli, and the generated enzymes were characterized. This study reveals the following. (i) For isozyme A, the C-terminal Tyr-363 and the N-terminal region bearing isozyme group-specific sequences 1-3 and Lys-107 (the C-6 phosphate-binding site) are responsible for the higher catalytic activity toward fructose 1,6-bisphosphate, which is 7 times higher than that of aldolase B. Conversely, an internal region spanning positions 108-212 is required for the lower activity toward fructose 1-phosphate. (ii) For isozyme B, an internal sequence spanning positions 108-212 which includes some isozyme B-specific residues and a postulated C-1 phosphate-binding site (Lys-146 or Arg-148) is responsible for a higher catalytic activity toward fructose 1-phosphate, which is 8-10 times that of isozyme A. The more upstream sequence containing positions 1-107 is responsible for the lower catalytic activity toward fructose 1,6-bisphosphate. (iii) At least residues 212-306, composing a long stretch near the active-site Lys-229 and highly conserved among isozymes A, B, and C, may be required for the basal framework of the aldolase molecule to exhibit the activity common to the three isozymic forms.

Amino Acid Sequence↗
PubMed1990

Nonconservative utilization of aldolase A alternative promoters.

Recently, analysis of the sequence and expression of the human aldolase A gene revealed the unique arrangement of three tandem promoters and exons preceding a common coding sequence. A muscle-specific promoter (M) and two flanking widely used promoters (N and H) produce mRNA species which, in their mature forms, differ only in the sequence of their 5'-untranslated regions. We have isolated and investigated the expression of a mouse aldolase A gene. This mouse gene represents a functional gene by sequence analysis, recombinational screening, and by transfection into C2C12 cells. Although there is a high degree of sequence similarity between the mouse and the human gene in the region of the alternative first exons, we have been unable to detect a functional utilization of the 5'-most promoter (N) in the mouse. Steady state mRNAs isolated from a variety of adult tissues and cultured cells were analyzed by RNase protection and primer extension to identify first exon utilization. Consistent with previous reports, exon M is found only in skeletal muscle and exon H, the "housekeeping" exon, is utilized in every tissue where aldolase A is expressed. Under identical conditions we fail to see any evidence of the N exon. Therefore, although sequence homology exists between rodents and primates in the N region, the absence of selective pressure to preserve its primate pattern of expression may have resulted in functional promoter extinction.

Amino Acid Sequence↗
PubMed1990

[Immunohistological localization of human aldolase A in human neoplastic and non-neoplastic tissues].

The expression of human aldolase A in tissues of cancer patients was examined immunohistologically using 4 monoclonal antibodies (1A2, 3C5, 1D1 and 4C2) to human aldolase A. In frozen sections, but not in paraffin sections, tumor cells of 22 out of 30 patients and non-neoplastic cells of 11 out of 21 patients examined reacted with 1A2 irrespective of cancer types. Positive reactions were seen mostly in the nucleus and some in the cytoplasm. Only tumor cells in a few neoplastic tissues gave positive cytoplasmic reactions with 1D1 or 4C2. No positive reaction was observed with 3C5. Antigens cross-reactive with aldolase A may be expressed more frequently in tumor cells than in non-neoplastic cells of cancer patients.

Antibodies, Monoclonal↗
PubMed1990

[The effect of magnesium sulfate used for killing rabbits on aldolase activity in organs].

The effect of the system of mating and the way of killing on the aldolase activity in the tissue of rabbits was the task of this research work. The research work was realized in 201 New Zealand white rabbits aged 140 days, mated outbred and inbred. Two methods of killing were used: classical method, which was based on the stroke and break the spinal cord; and killing with the help of treating with an i/m infection of 10 per cent of magnesium sulphate (2 ml per 1 kg of weight). In blood serum and in liver homogenates, kidneys and dorsal muscle the aldolase activity was determined by the method of Bruns. No significant differences in the aldolase activity between outbred and inbred rabbits were found. Statistically high significant differences, conditioned by killing method, were stated in activity of enzyme.

Animals↗
PubMed1989

[Cleavage and synthesis of sialic acids with aldolase. NMR studies of stereochemistry, kinetics, and mechanisms].

1H-NMR spectroscopy was used to study cleavage and synthesis of N-acetyl- and N-glycoloyl-D-neuraminic acid by Clostridium perfringens aldolase. Whereas the alpha-anomers of Neu5Ac and Neu5Gc serve as substrate in the cleavage reaction, alpha-ManNAc and alpha-ManNGc are its primary products. The same alpha-anomers are needed by the aldolase for the synthesis of Neu5Ac and Neu5Gc. During the enzyme reaction in D2O both H-atoms at C-3 of Neu5Ac are exchanged by deuterium, H-3e reacting faster than H-3a. Rate constants and concentrations at equilibrium of reactants are temperature- and pH-dependent: The amount of Neu5Ac in equilibrium increases with decreasing temperature and increasing pH-value. Based on these results a mechanism of aldolase action is discussed.

Clostridium perfringens↗
PubMed1989

Opposite effects of alfa-actinin and of fructose 1,6-bisphosphate aldolase on the microfilament network. The role of orthophosphate revisited.

At pH 7.5, in the presence of 0.1 M KCl, 2 mM MgCl2 and 15 mM phosphate, the binding of 1 molecule of alfa-actinin for each strand of 1000 actin monomers doubles the apparent viscosity of an F-actin solution (12 microM as the monomer). Further binding of one molecule of aldolase for each strand of 280 actin monomers halves the apparent viscosity of the alfa-actinin-F-actin system without any desorption of alfa-actinin. The effect of aldolase is not hindered by the addition of 0.1 mM fructose 1,6-bisphosphate. It is shown that orthophosphate acts as a damper of the regulatory effect of fructose bisphosphate on the interaction between aldolase and microfilaments.

Actin Cytoskeleton↗
PubMedDate not supplied

[Retention of specific activity of muscle aldolase after its immobilization on odigos and odifil (ethylsulfo-activated agarose)].

The effect of the number of active groups of new affinity supports--odigose and odifil (ethylsulfo-activated agarose) on the retention of the specific activity of muscle aldolase was investigated. The active center of the enzyme includes lysine able to react with activated supports. The aldolase completely retained the specific activity after immobilization on the abovementioned relatively high-substituted supports, on which other enzymes, e.g. phosphorylase B, NAD-kinase from pigeon heart, were partially or completely inactivated. The aldolase was inactivated when being immobilized on more substituted supports. The enzyme specific activity completely retained if the high substituted supports were preliminary incubated at 37 degrees to destroy some diazo-groups.

Animals↗
PubMed1986

Characterization of the human aldolase B gene.

The structure of the human gene encoding the aldolase B isozyme has been determined, including the sequence of 14,887 base-pairs. The 5'- and 3'-ends have been determined by S1 mapping. There is a single gene for this enzyme in humans that was determined from the sequence and restriction enzyme digestions of genomic DNA. The gene is 14,500 base-pairs long containing nine exons. In addition, 924 and 208 base-pairs of the 5'- and 3'-flanking region, respectively, have been determined. There is a high degree of conservation of nucleic acid sequence between aldolase B genes of human, rat and chicken. The conservation extends to untranslated and flanking regions, and includes the derived protein structures. In the 5'-flanking region there are several sequence elements that are conserved in vertebrate aldolase B genes in addition to the T-A-T-A and C-C-A-A-T boxes. These sequences may be involved in the co-ordinate and tissue-specific control of expression of this gene. Several possible polymorphic sites were detected in the sequence of the human gene which may be useful for linkage mapping of the human genome and diagnostic analysis of alleles in families with hereditary fructose intolerance.

Animals↗
PubMedDate not supplied

[Structuro-functional characteristics of aldolase from rabbit muscles in diabetes].

The structural peculiarities of rabbit muscle aldolase accompanying enhancement of the aldolase activity in diabetes are described from the data of tryptophan phosphorescence at the room temperature and fluorescence polarization. It is shown that the pathology-concomitant conformational changes occur in both the hydrophobic part and NAD-binding site of the enzyme. The character of the structural changes in the hydrophobic part of the protein in diabetes and an increase in the enzymic activity are similar to that observed in normal aldolase after its interaction with NADH and are believed to be associated with the enhancement of the rigidity in the Trp-147 environment.

Animals↗
PubMed1987

Concentration and partitioning of intermediates in the fructose bisphosphate aldolase reaction. Comparison of the muscle and liver enzymes.

Chemical analysis of enzyme reaction intermediates has been used to compare the liver and muscle isozymes of rabbit aldolase at equilibrium and in their steady states to determine if they have properties that favor the direction of flow of glycolytic intermediates in their tissues of origin. For both enzymes at saturating concentrations of fructose 1,6-P2, the sum of intermediates in the steady state agreed with the total active enzyme calculated to be present. The two half-reactions, characterized by fructose 1,6-bisphosphate(Fru-P2):aldehyde exchange and DHAP:proton exchange were found to be of different importance in determining the rate of reaction with Fru-P2 with the liver enzyme being much more limited in the processing of DHAP. The chemical interconversions within each half-reaction are generally rapid compared with the release of products. The greater sensitivity of liver aldolase to inhibition by aldehydes in Fru-P2 cleavage seems to be a normal consequence of the higher level of the eneamine of DHAP in the forward steady state with the liver enzyme and probably should not be ascribed to a greater intrinsic affinity. An earlier report (Grazi, E., and Trombetta, G. (1979) Eur. J. Biochem. 100, 197-202) purporting to show a special interaction of glyceraldehyde-3-P with liver enzyme prior to proton abstraction from DHAP could not be reproduced. Examples are presented from the data that validate the use of the analytical methods used for analysis of intermediates in the case of the Schiff's base aldolases.

Animals↗
PubMed1984

Nucleotide sequence of rat liver aldolase B messenger RNA.

The nucleotide sequence of messenger RNA encoding rat liver aldolase B has been determined by sequence analysis using recombinant cDNAs cloned in bacterial plasmids. The sequence contains part of the 5'-untranslatable region (68 nucleotides), the entire coding region (1092 nucleotides), and the complete 3'-untranslatable region (387 nucleotides), excluding the poly(A) tail. A potential ribosomal-binding site is located about 30 nucleotides upstream from the initiation codon. The amino acid sequence of rat liver aldolase B is composed of 364 amino acids and has 70% homology with rabbit muscle aldolase A.

Amino Acid Sequence↗
PubMed1980

Immunolocalization of fetal aldolase isoenzymes in rat regenerating liver after carbon tetrachloride intoxication.

Light and electron microscopic immunolocalization of adult and fetal aldolase isoenzymes has been studied in rat liver 72 h after CCL4 intoxication. As in rat regenerating liver after partial hepatectomy, fetal aldolases A and C were located in sinusoidal cells (Kupffer and endothelial cells) whereas adult aldolase B was present in hepatocytes only. Our results are different from those obtained with fetal and cancerous livers; they suggest that control mechanisms of gene regulation different in liver regeneration and in cancer.

Animals↗
PubMed1983

Aldolase release rate and platelet function.

We have developed a new platelet function test which is a method to estimate the rate of release of aldolase from platelets into a low osmotic pressured artificial medium and into PRP, which we called the platelet aldolase release rate. As this aldolase release rate have a good reproducibility and correlate well with other platelet function test, this test can be utilized for clinical examination.

Blood Platelets↗
PubMed1984

Association of phosphofructokinase and aldolase with the membrane of the intact erythrocyte.

The binding of phosphofructokinase and aldolase to the membrane of the intact human erythrocyte was assessed by the rapid hemolysis/filtration method of Kliman and Steck (Kliman, H. J., and Steck, T. L. (1980) J. Biol. Chem. 255, 6314-6321). We found that about 50% of the phosphofructokinase was membrane-bound in fresh red cells prior to hemolysis. Binding was not significantly altered by deoxygenation. Approximately 40% of aldolase was membrane-associated in fresh red cells. In outdated, blood-banked red cells, aldolase was 73% membrane-bound while, following metabolic repletion, 40% of the enzyme was membrane-associated. These results support the hypothesis that certain glycolytic enzymes in the red cell are membrane-bound in a rapidly reversible and metabolically sensitive fashion.

Erythrocyte Membrane↗
PubMed1984

[Increase in serum aldolase caused by bronchial carcinoma].

13 patients were undergone a resection of their lung tumours. Under operation aldolase were determined in V. pulmonalis, A. pulmonalis and V. basilica. First time it is demonstrated: Human bronchial carcinomas give aldolase in circulating blood. Aldolase-level in V. pulmonalis of carcinoma increases with size and dedifferentiation of tumours.

Arm↗