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)

Human erythrocyte ghosts were solubilized in a low ionic strength medium containing 1% Triton X-100 and subjected to electrophoresis in polyacrylamide gels containing Triton X-100. Five major bands were stained with Coomassie Blue, all except one band being heterogenous when re-electrophoresed in gels containing sodium dodecyl sulphate. It was possible to detect acetylcholinesterase, non-specific esterase, ATPase, alkaline phosphatase, 5'-nucleotidase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aldolase activities on the Triton-containing polyacrylamide gels. Two of the enzymes, ATPase and 5'-nucleotidase, showed substantial inhibition by Triton X-100 in quantitative studies. This appears to be a useful method for studying membrane enzymes in normal and pathological red cells.
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PMID:Polyacrylamide gel electrophoresis of human erythrocyte membrane enzymes solubilized with triton X-100. 89 Sep 65

Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD.
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PMID:Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane. 125 46

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

Distribution of several glycolytic enzymes in the lenses of different vertebrate species and their organization in the calf lenses were studied. Though no general pattern of enzyme activities in different species is discernible, high activities of TPI followed, in decreasing order, by GAPDH, enolase, PK, LDH and aldolase appear to be more common. Our observation on the unusually high activities of aldolase in the pig, enolase in the sheep and LDH in the duck lens are interesting in view of the already known dual function of LDH as an enzyme and a structural protein (epsilon-crystallin) in duck. Controlled treatment with detergents Brij-58 and Triton X-100 caused distinctly differential purturbations in the lens cells. In spite of fiber membrane disruption and partial actin dissolution by Brij-58, no significant increase in the release of glycolytic enzymes compared to control was observed. This suggests that none of the enzymes existed as a completely soluble and freely diffusible fraction. But treatment with a strong detergent (Triton X-100) caused the release of higher amounts of enzymes suggesting either a direct or indirect interaction with the cytomatrix components. Aldolase appears to be maximally bound in the cytosol followed by TPI, GAPDH, LDH and PK in decreasing order. Although thin lens slices were incubated with the detergents for a total period of 40 min and the loss of fiber architecture and organization confirmed by light microscopy, in the Triton X-100 treated tissues less than 25% of the total activity of any enzyme except TPI appeared in the bathing medium.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Investigation of lens glycolytic enzymes: species distribution and interaction with supramolecular order. 155 53

The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.
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PMID:Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle. 191 47

When assayed in vitro, the activity of the photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase oxygenase is both enhanced and protected from spontaneous decay by exogenous proteins such as hemoglobin, serum albumin, and aldolase. Other proteins and amino acids tested are either ineffective (lysozyme, ferritin, lysine, and cysteine) or afford only partial protection (catalase, glycine, and phenylalanine). Protective proteins do not bind to, or exchange disulfides with, ribulose 1.5 bisphosphate carboxylase/oxygenase. Since their effect can be mimicked by reductively treated detergents such as Triton X-100, it appears that proteins protect from decay by quenching the spontaneous oxidative degradation and inhibiting surface adsorption which could lead to enzyme unfolding. Release of adsorbed molecules from the container surface is likely to be the cause of carboxylase activity enhancement.
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PMID:Protection and enhancement of ribulose 1,5 bisphosphate carboxylase activity by exogenous proteins. 191 Apr 60

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.
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PMID:Trifluoperazine activates and releases latent ATP-generating enzymes associated with the synaptic plasma membrane. 358 33

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.
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PMID:Cross-linking of the enzymes in the glycosome of Trypanosoma brucei. 399 56

Detergent extraction of brain slices and mouse fibroblast 3T3 cells was performed to determine rates and relative amounts of extraction of inositol versus the glycolytic enzymes. The two detergents, Triton X-100 and Brij 58, led to similar results for extraction of myo-inositol. The extraction of enzymes from brain slices or cells varied with the detergent. In brain slices, a buffered solution containing 0.2% of the detergent Brij 58 led to the extraction of 85% of the inositol before 3% of the aldolase or before 37% of either lactate dehydrogenase or triose phosphate isomerase was extracted. In contrast, with 0.1% Triton X-100 in isotonic phosphate-buffered saline, when 70% of the inositol was extracted, 33% of the aldolase and 48% of the triose phosphate isomerase were extracted. Lesser amounts of aldolase and glyceraldehyde phosphate dehydrogenase were extracted than most of the other glycolytic enzymes under all conditions, implying that these enzymes may be interacting with non-extractable subcellular components. In 3T3 cells, both detergents were of similar effectiveness for inositol extraction. Triton X-100 caused 89% of the inositol to be released and Brij 58 caused 84% to be released. With the enzymes, Brij 58 caused between 15 and 38% extraction and Triton X-100 caused between 61 and 85% extraction of the different glycolytic enzymes. Thus Brij 58 was as effective as Triton X-100 in inositol extraction but not nearly as effective in glycolytic enzyme extraction. The results demonstrate that inositol leakage from tissues or cells is a better indicator of detergent-mediated alterations in membrane porosity than glycolytic enzyme leakage.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Extraction of glycolytic enzymes: myo-inositol as a marker of membrane porosity. 404 57

The nature of the association of the glycolytic enzyme, aldolase, with mature bovine spermatozoa was investigated in comparison with bovine muscle aldolase. Bovine muscle aldolase (BMA) was optimally solubilized by 0.1% deoxycholate and purified to homogeneity by ammonium sulfate fractionation, gel-filtration chromatography and phosphocellulose affinity chromatography. Bovine sperm aldolase (BSpA) was solubilized with optimal specific activity by 0.1% Triton X-100 and 50 mM sodium phosphate. Soluble BSpA represented 10% of the total aldolase activity in bovine spermatozoa. It could not be purified from other sperm components by standard procedures. The association of BSpA with sperm components involved noncovalent, ionic and hydrophobic interactions and did not involve disulfide bonds or covalent bonds. The stability of the BSpA association with intracellular substructure implies that very specific multiple-ligand bonding is involved. The Km for fructose-1-phosphate (1.7 X 10(-1) M) was higher and the activity with fructose-1,6-biphosphate relative to fructose-1-phosphate (Vmax FBP/Vmax F-1-P = 0.038) was much lower than for either liver or muscle aldolase. Kinetic analysis and subcellular associations indicated that sperm aldolase is different from other isozymes of aldolase.
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PMID:Association of bovine sperm aldolase with sperm subcellular components. 646 57


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