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)

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.
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PMID:Construction and properties of active chimeric enzymes between human aldolases A and B. Analysis of molecular regions which determine isozyme-specific functions. 221 42

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

Hereditary fructose intolerance (HFI) is an inborn error of metabolism, inherited as an autosomal recessive disorder and caused by a decrease in the activity of fructose-1-phosphate aldolase (aldolase B) in affected individuals. Investigation of the molecular basis of HFI is reported here by the identification of two molecular lesions in the aldolase B gene of the HFI individual. Using polymerase chain reaction to specifically amplify exons at this locus and T7 polymerase for the sequence determination of these double-stranded fragments, we show the mutational heterogeneity of the proband. One allele, previously indicated by restriction analysis, was confirmed as A149P (Ala 149 to Pro in exon 5). The other allele was identified as a 4-bp deletion found in exon 4, a deletion which causes a frameshift at codon 118, resulting in a truncated protein of 132 amino acids. Segregation of these mutant alleles in the proband's family was shown by using allele-specific oligodeoxynucleotides to probe blots of amplified DNA. The techniques employed here represent a rapid and efficient method for detection of other mutations in families with this disease. In addition, the ability to detect mutant alleles by allele-specific hybridization offers a new method for definitive diagnosis, a method which avoids a fructose loading or liver-biopsy examination.
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PMID:Molecular evidence for compound heterozygosity in hereditary fructose intolerance. 233 10

An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U-13C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13C NMR spectra of plasma glucose. The conversion parameters in the control and HFI children were estimated on the basis of doublet/singlet values of the plasma beta-glucose C-1 splitting pattern as a function of the rate of fructose infusion (0.26-0.5 mg/kg per min). Significantly lower values (approximately 3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13C NMR measurement of plasma [13C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13C atoms at glucose C-4 (13C3-13C4-13C5) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only approximately 50% of the total amount of hepatic fructose conversion to glucose. It is suggested that phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate by 1-phosphofructokinase occurs in human liver (and intestine) when fructose is administered nasogastrically; 47% and 27% of the total fructose conversion to glucose in controls and in HFI children, respectively, takes place by way of this pathway. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [13C]glucose formation from a "trace" amount (approximately 20 mg/kg) of [U-13C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.
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PMID:Determination of fructose metabolic pathways in normal and fructose-intolerant children: a 13C NMR study using [U-13C]fructose. 237 Dec 80

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

A highly sensitive enzyme immunoassay system for measurement of aldolase B subunit (aldolase B) was established. Antisera were raised in rabbits by injecting aldolase B4 purified from human liver, and specific antibodies to aldolase B were purified by the use of a column of aldolase B4-coupled Sepharose. The purified antibody IgG was digested with pepsin to obtain the F(ab)' fragments. The antibody F(ab)' fragments were immobilized noncovalently on polystyrene balls, and the same antibody Fab' fragments were labeled with beta-D-galactosidase from Escherichia coli. The sandwich-type assay system using these reagents was sensitive and specific to aldolase B, showing no cross-reactivity with aldolase-A or aldolase-C. The minimum detection limit of the assay was 3 pg aldolase B4/assay tube. The immunoreactive aldolase B was present at high levels in the liver and kidney, and considerably in the small intestine. It was detected in all the tissues examined. Immunohistochemically, aldolase B is localized in hepatocytes, proximal renal tubular cells and epithelial cells of small intestine. Serum levels of aldolase B in healthy subjects were ranged from 33 to 202 ng/ml.
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PMID:Sensitive enzyme immunoassay for human aldolase B. 265 6

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

Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and greater than 30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, greater than 30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the greater than 30-kb fragment and is probably localized on chromosome 3 with the greater than 30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and greater than 30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes; the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.
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PMID:Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10. 282 24


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