Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The site-specific modification of rabbit muscle aldolase A by labeling of thiol residues of Cys-289 with 5-(2-((iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid and Cys-239 with 5-iodoacetamidofluorescein or 4-dimethylamino-phenylazophenyl-4'-maleimide has been described. The method is based on the differences in kinetics of the chemical modification of aldolase thiols with the above reagents either in the presence or in the absence of a competitive inhibitor. The spectral properties of the doubly labeled aldolase derivatives were compared with those of the singly labeled enzyme. The doubly labeled aldolase derivatives exhibited full catalytic activity.
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PMID:Site-specific modification or rabbit muscle aldolase with fluorescent probes. 212 52

A sensitive sandwich-type enzyme immunoassay for the aldolase isozyme, A4, was developed using purified antibodies specific to the A subunit of aldolase. The antibodies were raised in sheep being immunized with purified aldolase A4 and then purified by immunoaffinity chromatography on a column of aldolase A4-coupled Sepharose. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody F(ab')2 fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was sensitive enough to detect 10 pg/tube of aldolase A4. The assay was specific to the A subunit of aldolase (aldolase A). It cross-reacted about 40% to aldolase A3C, 7% to A2C2 and 0.3% to AC3, but not cross-reacted with C4 nor B4. Coefficients of variation in intra- and inter-assay were less than 16%. Serum aldolase A levels were determined in healthy adults, which were about 200 ng/ml. The distribution and concentrations of immunoreactive aldolase A in various human tissues were also determined. High concentrations of aldolase A were found in skeletal muscle, heart muscle, cerebrum and lymphatic tissue.
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PMID:Sensitive enzyme immunoassay for human aldolase A. 218 23

Enzymatic studies on aldolase isozymes have been carried out by techniques of protein engineering. Site-directed mutagenesis helps us to verify the roles of amino acid residues in catalytic reactions. Chimeric fusion proteins give us information about the regions which specify the characteristics of the isozymes. The results are: (1) In aldolase A, COOH terminal Tyr and Lys-107 residues play important roles in catalysis, especially in binding of FDP. (2) Aspartic acid at the 128th residue in aldolase A is essential to thermostability; no other residue such as glutamic acid can substitute for it. (3) Studies on chimeric fusion proteins indicate that the C-terminal region (including C-terminus Tyr) or aldolase A is responsible for its substrate specificity, which is not seen in aldolase B. (4) A region near NH2 terminus in aldolase B determines its specific structure. (5) The region including His-107, Asp-128, and Tyr-137 (B-A junction of BA137) is located in a turn which is exposed outward (a model architecture by Sygusch et al [1987]). In BA137, this region would be constrained, and play a significant role in catalysis, thermostability, etc. (6) Tertiary structure of aldolase B seems to be dissimilar to that of aldolase A.
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PMID:Structural studies on aldolase isozymes through protein engineering. 220 67

A subunit specific radioimmunoassay was developed for the quantification of human aldolase A, B, and C. The method used was a double antibody radioimmunoassay using radioiodinated purified aldolase A, B, or C subunits as the ligand, specific chicken antibodies to aldolase isozymes and rabbit antibodies to chicken IgG. The Iodogen method was used for iodination of the purified isozyme subunits in this study. Human brain tissue contained similar concentrations of aldolase A and aldolase C, and a smaller amount of aldolase B, which was the main isozyme of liver tissue. Levels of serum aldolase A were greater than 203 ng/ml, the upper limit of normal, in six of 24 patients with cerebral infarction and in 11 of 31 patients with cerebral hemorrhage. Nine of 24 patients with cerebral infarction and 16 of 31 patients with cerebral hemorrhage had serum aldolase C levels greater than 4.1 ng/ml, the upper limit in normal sera. These data suggest that serum aldolase C may be a more specific and sensitive marker of cerebrovascular diseases than aldolase A. We also demonstrated that serial measurement of serum aldolase C in patients with cerebrovascular diseases might be useful in estimating prognosis, since serially increasing serum aldolase C levels during the course of these diseases were correlated with a high mortality rate.
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PMID:Serum aldolase isozyme levels in patients with cerebrovascular diseases. 224 17

Aldolase A, B, and C were determined in rat liver and serum by radioimmunoassay (RIA) in order to evaluate the alteration of these isozymes in the process of hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), and the immunohistochemical technique was also used for the analysis of localization of aldolase isozymes. Aldolase A was increased in cancer tissues of 3'-Me-DAB induced hepatoma, whereas aldolase B was decreased in the same tissues according to both RIA and the immunohistochemical technique. During the promotion stage of hepatocarcinogenesis, the cells in hyperplastic nodules, which are known as preneoplastic lesions, were stained for aldolase A. Aldolase C was slightly increased in cancer tissues by RIA, suggesting the increase of A-C hybrid like A3C which was demonstrated by the electrophoretic method. Serum aldolase A levels were not significantly elevated in rats with liver cancer in comparison to rats with non-cancer.
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PMID:[Biochemical and immunohistochemical studies on alteration of aldolase isozymes in rat liver in the process of hepatocarcinogenesis by administration of a diet containing 3'-methyl-4-dimethylaminoazobenzene]. 251 Nov 29

The structure of the chromosomal gene encoding rat aldolase isozyme B has been elucidated by sequence analysis of cloned genomic DNA. This gene comprises about 14 X 10(3) base-pairs of DNA, and is separated into nine exons by eight intervening sequences. A presumed transcription-initiation site was assigned by S1 nuclease protection mapping, and T-A-T-A and C-C-A-A-T boxes were found to be 25 and 126 base-pairs, respectively, upstream from this initiation site. There are three characteristic sequences of 100 to 200 base-pairs within the region of 870 base-pairs flanking the 5' side of the gene. These sequences are flanked on either side by direct repeats and terminate with an A-rich stretch of nucleotides. One of them has block homology with a region in an "ID sequence", which is reported to be an element for tissue-specific gene regulation and differentiation. The other two are analogous at the sequence organizational level with a sort of dispersed repeat, the "Alu family". These features suggest that these regions are involved in gene regulation and, also, imply evolutionary events such as duplication or insertion. Comparison of this gene sequence with the rabbit aldolase A complementary DNA sequence revealed some bias in the frequency of nucleotide replacement among the exons, suggesting selective evolutionary conservation of particular exons encoding functional domains. Comparison with the human aldolase B complementary DNA sequence revealed no such tendency; the homology between the two sequences was very high (about 89%), and nucleotide replacements were randomly distributed throughout the protein-coding region.
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PMID:Structure and genomic organization of the rat aldolase B gene. 258 98

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.
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PMID:Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host. 264 52

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)
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PMID: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. 265 66

The partition behaviour of six enzymes of the Calvin cycle in extracts of chloroplasts from spinach (Spinacia oleracea) between two aqueous phases has been studied by countercurrent distribution. The enzymes showed distribution patterns which indicate heterogeneity and the presence of two or three fractions of most of the enzymes. When two of the enzymes, phosphoglycerate kinase and fructose-bisphosphate aldolase, were partitioned in both purified and partially purified form, they behaved like homogeneous substances. These results indicate that countercurrent distribution of crude extracts in aqueous two-phase systems is a useful method to study protein-protein interaction.
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PMID:Studies of protein-protein interaction using countercurrent distribution in aqueous two-phase systems. Partition behaviour of six Calvin-cycle enzymes from a crude spinach (Spinacia oleracea) chloroplast extract. 273 May 89

Fructose-1,6-bisphosphate aldolase A (fructose-bisphosphate aldolase; EC 4.1.2.13) deficiency is an autosomal recessive disorder associated with hereditary hemolytic anemia. To clarify the molecular mechanism of the deficiency at the nucleotide level, we have cloned aldolase A cDNA from a patient's poly(A)+ RNA that was expressed in cultured lymphoblastoid cells. Nucleotide analysis of the patient's aldolase A cDNA showed a substitution of a single nucleotide (adenine to guanine) at position 386 in a coding region. As a result, the 128th amino acid, aspartic acid, was replaced with glycine (GAT to GGT). Furthermore, change of the second letter of the aspartic acid codon extinguished a F ok I restriction site (GGATG to GGGTG). Southern blot analysis of the genomic DNA showed the patient carried a homozygous mutation inherited from his parents. When compared with normal human aldolase A, the patient's enzyme from erythrocytes and from cultured lymphoblastoid cells was found to be highly thermolabile, suggesting that this mutation causes a functional defect of the enzyme. To further examine this possibility, the thermal stability of aldolase A of the patient and of a normal control, expressed in Escherichia coli using expression plasmids, was determined. The results of E. coli expression of the mutated aldolase A enzyme confirmed the thermolabile nature of the abnormal enzyme. The Asp-128 is conserved in aldolase A, B, and C of eukaryotes, including an insect, Drosophila, suggesting that the Asp-128 of the aldolase A protein is likely to be an amino acid residue with a crucial role in maintaining the correct spatial structure or in performing the catalytic function of the enzyme.
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PMID:Human aldolase A deficiency associated with a hemolytic anemia: thermolabile aldolase due to a single base mutation. 282 99


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