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)

Fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phyosphate-lyase, EC 4.1.2.13) was isolated from buffalo muscle by fractionation with ammonium sulphate and subsequent purification by phosphocellulose column chromatography using a linear salt gradient. As judged by gel filtration and electrophoresis in polyacrylamide gel, the enzyme was homogeneous with respect to size and charge. The molecular weight and Stokes radius of the enzyme were determined from its elution profile on a calibrated Sephadex column and the respective values were 162000 and 4.55 nm. The diffusion coefficient and frictional ratio were computed to be 4.8-10(7) cm2-s-1 and 1.27, respectively. The molecular weight of the polypeptide chain as measured by aodium dodecyl sulphate polyacrylamide gel electrophoresis was 40750. This taken together with the native molecular weight suggested a four-subunit model for the protein. The N- AND C-terminal residues of polypeptide chains were identified to be proline and tyrosine, respectively. At pH 8.0 the Michaelis-Menten constant and maximum attainable velocity were found to be 8.1 muM and 27 muM Fru-1,6-P2 split/min per mg, respectively. The buffalo muscle aldolase was found to be similar to rabbit muscle aldolase in physico-chemical properties. However, the two enzymes differ significantly in pH optimum; the p optima of the buffalo and rabbit enzymes were determined under identical conditions to be 8.0 and 8.6, respectively.
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PMID:Isolation of buffalo muscle aldolase and comparison of its properties with those of rabbit muscle aldolase . 1 72

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.
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PMID:Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate. 18 Oct 51

A procedure has been developed for the purification of human erythrocyte aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.1.3). The process involves a specific substrate elution of the enzyme from phosphocellulose followed by a reverse ammonium sulfate fractionation. The preparation has been shown to be homogeneous by analytical ultracentrifugation, thin-layer electrophoresis, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme exhibits a specific activity of 16 I.U./mg protein, a Km of 7.1-10(-6) M for fructose 1,6-bisphosphate, and a substrate specificity (Fru-1,6-P2/Fru-1-P) of 40. The native protein in a tetramer of 158 000 molecular weight possessing identical or nearly identical subunits, an isoelectric point of 8.9, a diffusion coefficient of 4.68-10(-7) cm2/s, and a molecular radius of 4.56 nm. The study shows the enzyme to be a type A aldolase resembling other muscle forms in chemical and physical properties as well as amino acid composition.
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PMID:Purification and characterization of aldolase from human erythrocytes. 88 43

An X-ray crystallographic structure determination has been carried out on 2-keto-3-deoxy-6-phosphogluconic (KDPG) aldolase at 3.5-A resolution using the multiple isomorphous replacement method with three heavy atom derivatives along with anomalous dispersion contributions from two of the derivatives. Crystals grown from ammonium sulfate-phosphate buffered (pH 3.5) solutions were: cubic, a= 103.40 (4) A, space group P213. KDPG aldolase consists of trimeric heterologous assemblages utilizing crystallographic threefold symmetry. The overall profile of the oligomeric structure viewed down the threefold axis resembles that of a ship propeller while the subunits are approximate irregular oblate ellipsoids (25 X 45 X 45 A). The folding of most of the polypeptide chain was traced unambiguously. Secondary structural features consist of nine helical regions (75 residues, 35%) and a pair of two parallel chains. The subunit contains a long empty channel which is about 9 X 9 X 30 A with one of the pair of parallel chains forming part of the wall. Three mercury binding sites are located in this channel. These might correspond to the two readily accessible and one of the two buried cysteine residues of each subunit. The channel terminates with another cavity of about 8 X 10 X 25 A near the surface of the oligomeric structure. The regions of the subunits near the threefold axis are characterized by a high degree of secondary structural organization and these make close intersubunit contacts. Quarternary interactions are due mainly to side-chain interactions of helices.
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PMID:The folding and quaternary structure of trimeric 2-keto-3-deoxy-6-phosphogluconic aldolase at 3.5-A resolution. 97 67

Deoxyribose 5-phosphate aldolase was purified 41 times from Bacillus cereus induced by growth on deoxyribonucleosides. The purification procedure includes ammonium sulphate fractionation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and preparative electrophoresis on 10% polyacrylamide gel. The enzyme is stable above pH 6.5, but is rapidly inactivated by sulfhydryl reagents. Being insensitive to EDTA, it may be considered as a Class I aldolase. Among a number of compounds tested (including some carboxylic acids, free and phosphorylated pentoses, nucleotides and nucleosides), none has been found to affect the enzyme activity. The enzyme appears to be dimeric, with a subunit Mr of 23,600. A Km of 4.4 x 10(-4) M was calculated for dRib 5-P.
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PMID:Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties. 173 28

A method for a 50-60-fold purification of a cysteine proteinase from trophozoites of Entamoeba histolytica using 35-80% ammonium sulphate fractionation, gel chromatography on Sephadex G-75, and preparative isoelectric focusing is described. The enzyme was examined for its proteolytic potencies towards native enzyme substrates. The amebic proteinase directly inactivates aldolase and glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle as well as glucose-6-phosphate dehydrogenase from yeast. The inactivation of citrate synthase from porcine heart proceeds rather slowly, whereas malate dehydrogenase from porcine heart is not affected by the amebic proteinase under the condition used. With the exception of aldolase all inactivated enzyme substrates have been cleaved by limited proteolyses yielding major cleavage products. The inactivation of aldolase probably functions by the release of a small segment from a terminus being essential for aldolase activity.
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PMID:Cysteine proteinase of Entamoeba histolytica. I. Partial purification and action on different enzymes. 287 Apr 30

1. Aldolases were isolated from the ordinary muscle of red sea bream Pagrus major, Pacific mackerel Scomber japonicus, and carp Cyprinus carpio by ammonium sulfate fractionation, followed by ion-exchange chromatography on DEAE-cellulose and CM-Sepharose CL-6B columns, and examined for enzymatic properties. 2. The aldolases showed the highest activity in a pH range from 6.8-7.8 Km values for fructose-1,6-bisphosphate ranged from 0.025-0.10 mM. 3. Irrespective of fish species, aldolase activity was inhibited by ATP, ADP, and AMP. ATP showed the strongest inhibition and was competitive with fructose-1,6-bisphosphate. 4. The aldolases did not require divalent metal ions for activation and were completely inhibited at 0.1 mM Cu2+. 5. Thermal inactivation of the enzymes was of the first-order reaction. Red sea bream, Pacific mackerel and carp enzymes lost the activity by 50% when incubated at 50 degrees C for 8, 14 and 23 min, respectively.
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PMID:Enzymatic properties of fish muscle aldolase. 292 47

A rat brain S100-binding protein, R40,000, has been isolated, characterized, and identified as fructose-1,6-bisphosphate aldolase. R40,000 was purified by ammonium sulfate precipitation, hydroxylapatite chromatography, dye-binding chromatography, and electroelution from sodium dodecyl sulfate-polyacrylamide gels. Microsequence analysis of a fragment of R40,000 revealed a 15-residue amino acid sequence which shows a high degree of homology to the amino acid sequence of fructose-1,6-bisphosphate aldolase from rabbit muscle and rat liver. Further characterization demonstrated that R40,000 has an amino acid composition, subunit molecular weight, and cyanogen bromide map similar to aldolase. In addition, purified aldolase interacts with S100 alpha and S100 beta by gel overlay, and aldolase enzyme activity is stimulated 2-fold in vitro by S100 alpha and S100 beta. S100 interacts predominantly with the C or brain-specific form of the enzyme in gels and stimulates the activity of the C-enriched form of the enzyme in a calcium-dependent manner. Altogether, these data suggest that fructose-1,6-bisphosphate aldolase may be an intracellular target of S100 action in brain.
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PMID:Identification of a molecular target for the calcium-modulated protein S100. Fructose-1,6-bisphosphate aldolase. 373 59

A human hybridoma clone (4E3) has been established by fusing lymphocytes from a lymph node taken from a breast cancer patient and human lymphoblastoid cells, LICR-LON-HMy2, by the poly(ethylene glycol) method. 4E3 has been stabilized and continued to secrete IgMk antibody into culture medium (greater than 10 micrograms/ml) for over 1 year. The following characteristics of the antigen strongly suggested that 4E3 recognizes liver-type aldolase B (EC 4.1.2.13): the Mr of the native molecule is 160,000 and that of the subunit is 40,000, and thus it has a tetrameric structure of identical subunits; the antigen is abundant in the liver and kidney of human, mouse and rabbit, and is localized by immunohistochemical methods in the cytoplasm of hepatocytes and in the proximal tubules of the kidney; the antigen is precipitable by 50-80% saturation with (NH4)2SO4; the antigen shows charge-dependent heterogeneity on DEAE-cellulose chromatography. To confirm this notion, aldolase B was purified to homogeneity from the liver of human, mouse and rabbit by phosphocellulose chromatography. During the chromatographic purification, the antigen activity as assayed by enzyme-linked immunosorbent assay (e.l.i.s.a.) was superimposed on the enzymic activity of aldolase. Furthermore, monoclonal antibody 4E3 strongly reacted with purified aldolase B in SDS/polyacrylamide-gel electrophoresis followed by Western blotting and also in e.l.i.s.a. using microplates coated with purified enzyme. The reaction between aldolase B and 4E3 activated the human complement system as assessed by the attachment of C3 to the immune complex of aldolase B and 4E3.
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PMID:Human monoclonal antibody recognizing liver-type aldolase B. 382 73

Rabbit liver aldolase was purified by affinity elution on a CM52 ion exchanger. Crystals of rabbit liver aldolase suitable for X-ray diffraction experiments were grown from 45% saturated ammonium sulfate solution at 4 degrees C. The enzyme crystallizes in space group C222(1) having cell dimensions a = 377.02 A, b = 130.35 A, c = 80.04 A and diffracts to at least 3.5 A resolution. On the basis of a 55% solvent content there are eight aldolase tetramers in the unit cell. Rotational symmetry analysis to 6.7 A is consistent with the aldolase tetramers having a high degree of internal symmetry corresponding to point group 222. The crystallized enzyme is catalytically active.
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PMID:Preliminary crystallographic investigation of rabbit liver aldolase. 407


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