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)

This investigation was undertaken to develop cytotoxicity assay systems using primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate oculotoxic agents and the ability of these in vitro assay systems to predict irritancy potential and delayed toxicity. We have characterized the epithelial nature of the cultures by identifying keratins with antikeratin antibodies (AE1/AE3) and by demonstrating metabolic enzymes important to the integrity of the cells: lactate dehydrogenase, glucose 6-phosphate dehydrogenase and aldolase. Eight surfactants were compared and ranked according to their cytotoxic potential. We evaluated cytotoxicity by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium, by making morphological observations and by assessing lysosomal neutral red uptake and mitochondrial 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. The cells were treated for 1 h with the surfactants and the possibility of delayed toxicity was evaluated 24 h after removal of the surfactant. The cytotoxicity of the different types of surfactants as shown by all the tests was cationic > anionic = amphoteric > non-ionic. Triton X-100, a non-ionic surfactant but a severe irritant, had a ranking similar to anionic surfactants. The in vitro rankings corresponded well to reported in vivo Draize rabbit eye test data. The 24-h test for lactate dehydrogenase leakage showed that mild and non-irritating surfactants did not demonstrate any subsequent damage after a 1-h exposure, but the extreme and severe surfactants continued to show further damage after the 1-h exposure. These in vitro findings were similar to reported in vivo results. The neutral red and MTT tests did not adequately predict the prolonged toxicity of the more irritating surfactants, as was demonstrated by the lactate dehydrogenase leakage test. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to rank the cytotoxic potential of surfactants, but only the lactate dehydrogenase leakage test was able to assess prolonged cell injury.
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PMID:Evaluation of surfactant cytotoxicity potential by primary cultures of ocular tissues: I. Characterization of rabbit corneal epithelial cells and initial injury and delayed toxicity studies. 128 45

The major anion exchanger in type A intercalated cells of the cortical and medullary collecting ducts of the human kidney is a truncated isoform of erythrocyte band 3 (AE1) that lacks the N-terminal 65 residues. Because this missing sequence has been implicated in the binding of ankyrin, protein 4.1, several glycolytic enzymes, hemoglobin, and hemichromes in erythrocytes, we have undertaken examination of the structure and peripheral protein interactions of this kidney isoform. The cytoplasmic domain of kidney band 3, kidney CDB3, was expressed in Escherichia coli and purified to homogeneity. The kidney isoform exhibited a circular dichroism spectrum and Stokes radius similar to its larger erythrocyte counterpart. Kidney CDB3 was also observed to engage in the same conformational equilibrium characteristic of erythrocyte CDB3. In contrast, the tryptophan and cysteine clusters of kidney CDB3 behaved very differently from erythrocyte CDB3 in response to pH changes and oxidizing conditions. Furthermore, kidney CDB3 did not bind ankyrin, protein 4.1, or aldolase, and expression of erythrocyte CDB3 was toxic to its bacterial host, whereas expression of kidney CDB3 was not. Taken together, these data suggest that the absence of the N-terminal 65 amino acids in kidney CDB3 eliminates the major function currently ascribed to CDB3 in erythrocytes, i.e. that of peripheral protein binding. The primary function of residues 66-379 found in kidney CDB3 thus remains to be elucidated.
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PMID:Partial characterization of the cytoplasmic domain of human kidney band 3. 762 93

The N-terminal cytoplasmic domain of the anion exchanger 1 (AE1 or band 3) of the human erythrocyte associates with peripheral membrane proteins to regulate membrane-cytoskeleton interactions, with glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase and aldolase, with the protein-tyrosine kinase p72syk, with hemoglobin and with hemichromes. We have demonstrated that the N-terminal cytoplasmic domain of band 3 (CDB3) is a substrate of the apoptosis executioner caspase 3 (1). CDB3 has two non-conventional caspase 3 cleavage sites, TATD45 and EQGD205 (2). In vitro treatment of recombinant CDB3 with caspase 3 generated two fragments, which could be blocked by pretreatment with the caspase 3 inhibitor Z-DEVD-fmk (3). Recombinant CDB3 in which the caspase 3 cleavage sites Asp45 and Asp205 were mutated, was resistant to proteolysis (4). Proteolytically derived fragments crossreactive with polyclonal anti-band 3 antibody appeared with simultaneous cleavage of poly (ADP-ribose) polymerase and procaspase 3 in staurosporine (STS)-treated HEK293 cells transiently transfected with CDB3 (5). In vivo cleavage of CDB3 could be blocked by pretreatment of cells with Z-DEVD-fmk or in cells transfected with mutant CDB3 (D45A, D205A) (6). Co-transfection experiments showed that STS-mediated cleavage of CDB3 diminished its interaction with the N-terminal domain of protein 4.2, confirming that such cleavage interferes with the interaction of CDB3 with cytoskeletal proteins (7). Active caspase 3 was observed in aged red cells but not in young cells. This red cell caspase 3 could cleave band 3 present in inside-out vesicles prepared from young erythrocytes arguing in favor of a physiological role of caspase 3 in aged erythrocytes.
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PMID:Caspase 3-mediated proteolysis of the N-terminal cytoplasmic domain of the human erythroid anion exchanger 1 (band 3). 1457 Sep 14

To characterize the location of glycolytic enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH2 terminus of band 3. First, a mAb to residues 1-12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH2 terminus of band 3. Fourth, a large increase in the accessibility of enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.
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PMID:Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane. 1570 94