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)

Three isoforms of fructose-1,6-bisphosphate aldolase were found to bind specifically to the actin-containing filament of the cytoskeleton and to show tissue-specific binding patterns. Aldolase A (muscle type) bound more tightly to the skeletal muscle cytoskeleton among the three isozymes, while aldolase B (liver type) preferred the liver cytoskeleton to those of other tissues. The specific binding of aldolase A to the skeletal muscle cytoskeleton was inhibited strongly by the substrates fructose 1,6-bisphosphate and fructose 1-phosphate. Several mutant aldolases A were examined to identify the amino acid residues or regions that play a role in specific binding. Among the mutant aldolases tested, A-E34D, A-K41N, and A-Y363S exhibited remarkably reduced binding activities. Experiments using FITC-labeled enzymes and Rh-labeled phalloidin disclosed that aldolase A associated with the cytoskeleton. Specifically, when aldolase A was incubated with human fibroblast MRC-5 permeabilized with Triton X-100, aldolase A bound to the actin filaments in the stress fibers within the cell. Aldolase A reversibly inhibited the contraction of MRC-5 cells which usually occurred in the presence of Mg2(+)-ATP and Ca2+. These results provide direct evidence that aldolase binds specifically to the actin-containing stress fibers and suggest that aldolase may regulate cell contraction through its reversible binding to the filaments in the permeabilized MRC-5 fibroblast.
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PMID:Mode of interactions of human aldolase isozymes with cytoskeletons. 924 96

Aldolase B is a peripheral membrane protein. Immobilized artificial membrane (IAM) surfaces were used to purify rat liver aldolase B in a single chromatographic step. Selective elution required dipalmitoylphosphatidylcholine (DPPC) to be included in the mobile phase. Selective elution of aldolase from the IAM column when DPPC (0.2 mM) was added to the mobile phase indicates that DPPC was an affinity displacing ligand for this membrane associated protein. Since tissue preparation involved only homogenization and centrifugation, the single step purification of aldolase B using IAM chromatography is a very convenient method. The IAM stationary phase (1.5 g) has a loading capacity of at least 4.39 mg total protein from rat liver homogenates and typically approximately 17.7 microg of pure aldolase in a single step from approximately 60 mg wet weight rat liver cytosol can be obtained.
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PMID:Single step purification of rat liver aldolase using immobilized artificial membrane chromatography. 944 62

Hereditary fructose intolerance (HFI, OMIM 22960), caused by catalytic deficiency of aldolase B (fructose-1,6-bisphosphate aldolase, EC 4.1.2.13), is a recessively inherited condition in which affected homozygotes develop hypoglycaemic and severe abdominal symptoms after taking foods containing fructose and cognate sugars. Continued ingestion of noxious sugars leads to hepatic and renal injury and growth retardation; parenteral administration of fructose or sorbitol may be fatal. Direct detection of a few mutations in the human aldolase B gene on chromosome 9q facilitates the genetic diagnosis of HFI in many symptomatic patients. The severity of the disease phenotype appears to be independent of the nature of the aldolase B gene mutations so far identified. It appears that hitherto there has been little, if any, selection against mutant aldolase B alleles in the population: in the UK, approximately 1.3% of neonates harbour one copy of the prevalent A149P disease allele. The ascendance of sugar as a major dietary nutrient, especially in western societies, may account for the increasing recognition of HFI as a nutritional disease and has shown the prevalence of mutant aldolase B genes in the general population. The severity of clinical expression correlates well with the immediate nutritional environment, age, culture, and eating habits of affected subjects. Here we review the biochemical, genetic, and molecular basis of human aldolase B deficiency in HFI, a disorder which responds to dietary therapy and in which the principal manifestations of disease are thus preventable.
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PMID:Hereditary fructose intolerance. 961 Jul 97

To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C. Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K. Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli. J Biochem (Tokyo) 1994;115:1172-7). Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K. Isozyme-specific modules on human aldolase A molecule. J Biol Chem 1993;268:1677-83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K. Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence. Prot. Eng. 1994;7:1387-93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C. The kinetic data also suggests that the connector-2 (amino acid residues 243-306) may modulate the interaction of IGS units with the alpha/beta barrel of the aldolase molecule.
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PMID:Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C. 985 14

A full-length clone of the aldolase B gene has been isolated from a cDNA library constructed from liver of Atlantic salmon (Salmo salar). Sequencing showed that the clone encodes a typical aldolase B, possessing a number of amino acid residues which are seen in aldolase B, but not in other aldolase isoforms. RT-PCR analysis showed that the gene is expressed in liver, kidney and intestine as expected. However, in contrast to mammalian and avian aldolase B, expression was also found in a number of other tissues. Levels of aldolase B mRNA in liver and kidney were not significantly altered during smoltification, the transformation of freshwater-dwelling salmon (parr) into saltwater-adapted salmon (smolts).
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PMID:Cloning and unusual expression profile of the aldolase B gene from Atlantic salmon. 987 39

We have previously shown that stress-induced protein degradation requires a functional ubiquitin-activating enzyme and the autophagic-lysosomal pathway. In this study, we examined the occurrence of ubiquitin-protein conjugates that form during nutrient starvation. Kidney and liver epithelial cells respond to nutrient stress by enhancing autophagy and protein degradation. We have shown that this degradative response was more dramatic in nondividing cultures. In addition, the onset of autophagy was suppressed by pactamycin, cycloheximide, and puromycin. We observed an accumulation of ubiquitinated proteins coincident with the degradative response to amino acid starvation. The stress-induced protein ubiquitination was not affected by cycloheximide, indicating that protein synthesis was not required. The ubiquitinated proteins were localized to the cytosol and subcellular fractions enriched with autophagosomes and lysosomes. The incorporation of the ubiquitinated proteins into autolysosomes was dramatically reduced by 3-methyladenine, an inhibitor of autophagy. The evidence suggests that ubiquitinated proteins are sequestered by autophagy for degradation. We next set out to identify those primary ubiquitinated proteins at 60 kDa and 68 kDa. Polyclonal antibodies were prepared against these proteins that had been immunopurified from rat liver lysosomes. The antibodies prepared against those 68 kDa proteins also recognized a 40 kDa protein in cytosolic fractions. Internal amino acid sequences obtained from two cyanogen bromide fragments of this 40 kDa protein were shown to be identical to sequences in liver fructose1,6-bisphosphate aldolase B. Anti-Ub68 antibodies recognized purified aldolase A and aldolase B. Conversely, antibodies prepared against aldolase B recognized the 40 kDa aldolase as well as four to five high molecular weight forms, including a 68 kDa protein. Finally, we have shown that the degradation of aldolase B was enhanced during amino acid and serum starvation. This degradation was suppressed by chloroquine and 3-methyladenine, suggesting that aldolase B was being degraded within autolysosomes. We propose that aldolase B is ubiquitinated within the cytosol and then transported into autophagosomes and autolysosomes for degradation during nutrient stress.
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PMID:Ubiquitinated aldolase B accumulates during starvation-induced lysosomal proteolysis. 988 86

Hereditary fructose intolerance (HFI) is a rare autosomal recessive, metabolic disorder, that results from a deficiency of aldolase B (fructose-biphosphate aldolase) in the liver, kidney and intestine. Recent molecular studies have identified the mutation A149P in most European patients. We describe the first case of HFI with molecular analysis in a Portuguese child, presenting the same mutation of the aldolase B gene. The role of molecular studies in the diagnosis of HFI risk patients and their families is emphasized.
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PMID:[Hereditary fructose intolerance]. 1019 89

A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with hereditary fructose intolerance, the offspring of a consanguineous union, has identified the novel mutation Ala337-->Val in homozygous form. This mutation was also detected independently in two other affected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala149-->Pro, indicating that the mutation causes aldolase B deficiency. To test for the effect of the mutation, catalytically active wild-type human aldolase B and the Val337 variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 microM and 2.4 mM respectively. The specific activities of purified tetrameric Val337 aldolase B, which affects an invariant residue in the C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 microM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val337 variant enzymes respectively. The Val337 mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val337 protein had a distinct thermal denaturation profile with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinity and activity towards its specific F-1-P substrate and maintained appreciable activity towards FBP. In contrast, fluorescence studies of the mutant showed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These findings in a rare but widespread naturally occurring mutant implicate the C-terminus in the activity of human aldolase B towards its specific substrates and demonstrate its role in maintaining the overall stability of the enzyme tetramer.
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PMID:Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337-->Val) in fructose intolerance. 1022 88

Hereditary fructose intolerance (HFI) is an autosomal recessive human disease that results from the deficiency of the hepatic aldolase isoenzyme. Affected individuals will succumb to the disease unless it is readily diagnosed and fructose eliminated from the diet. Simple and non-invasive diagnosis is now possible by direct DNA analysis that scans for known and unknown mutations. Using a combination of several PCR-based methods (restriction enzyme digestion, allele specific oligonucleotide hybridisation, single strand conformation analysis and direct sequencing) we identified a novel six-nucleotide deletion in exon 6 of the aldolase B gene (delta 6ex6) that leads to the elimination of two amino acid residues (Leu182 and Val183) leaving the message inframe. The three-dimensional structural alterations induced in the enzyme by delta 6ex6 have been elucidated by molecular graphics analysis using the crystal structure of the rabbit muscle aldolase as reference model. These studies showed that the elimination of Leu182 and Val183 perturbs the correct orientation of adjacent catalytic residues such as Lys146 and Glu187.
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PMID:Novel six-nucleotide deletion in the hepatic fructose-1,6-bisphosphate aldolase gene in a patient with hereditary fructose intolerance and enzyme structure-function implications. 1035 30

Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyze the reversible cleavage of phosphofructose esters into cognate triose (phosphates). Three vertebrate isozymes of Class I aldolase have arisen by gene duplication and display distinct activity profiles with fructose 1,6-bisphosphate and with fructose 1-phosphate. We describe the biochemical and biophysical characterization of seven natural human aldolase B variants, identified in patients suffering from hereditary fructose intolerance and expressed as recombinant proteins in E. coli, from which they were purified to homogeneity. The mutant aldolases were all missense variants and could be classified into two principal groups: catalytic mutants, with retained tetrameric structure but altered kinetic properties (W147R, R303W, and A337V), and structural mutants, in which the homotetramers readily dissociate into subunits with greatly impaired enzymatic activity (A149P, A174D, L256P, and N334K). Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for maintaining its full catalytic function.
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PMID:Expression, purification, and characterization of natural mutants of human aldolase B. Role of quaternary structure in catalysis. 1062 57


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