What controls glycolysis in bloodstream form Trypanosoma brucei?
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On the basis of the experimentally determined kinetic properties of the trypanosomal enzymes, the question is addressed of which step limits the glycolytic flux in bloodstream form Trypanosoma brucei. There appeared to be no single answer; in the physiological range, control shifted between the glucose transporter on the one hand and aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glycerol-3-phosphate dehydrogenase (GDH) on the other hand. The other kinases, which are often thought to control glycolysis, exerted little control; so did the utilization of ATP. We identified potential targets for anti-trypanosomal drugs by calculating which steps need the least inhibition to achieve a certain inhibition of the glycolytic flux in these parasites. The glucose transporter appeared to be the most promising target, followed by ALD, GDH, GAPDH, and PGK. By contrast, in erythrocytes more than 95% deficiencies of PGK, GAPDH, or ALD did not cause any clinical symptoms (Schuster, R. and Holzhutter, H.-G. (1995) Eur. J. Biochem. 229, 403-418). Therefore, the selectivity of drugs inhibiting these enzymes may be much higher than expected from their molecular effects alone. Quite unexpectedly, trypanosomes seem to possess a substantial overcapacity of hexokinase, phosphofructokinase, and pyruvate kinase, making these "irreversible" enzymes mediocre drug targets. (+info)
Mechanism of metabolite transfer in coupled two-enzyme reactions involving aldolase.
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Transient-state kinetic experiments and analyses have been performed to examine the validity of hitherto unchallenged evidence proposed to be indicative of a channelled transfer of triose phosphates from aldolase to glyceraldehyde-3-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase. The results lend no support to such proposals, but show that the kinetic behaviour of the examined aldolase-dehydrogenase reactions is fully consistent with a free-diffusion mechanism of metabolite transfer. (+info)
A functional arginine residue in rabbit-muscle aldolase.
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Rabbit muscle aldolase is irreversibly modified by the arginine-selective alpha-dicarbonyl, phenylglyoxal, loss of activity correlating with the unique modifications of one arginine residue per subunit, as determined by amino acid analysis, and (7-14C)phenylglyoxal incorporation. The affinity of the modified enzyme for dihydroxyacetone phosphate is significantly reduced while substantial protection against inactivation is afforded by fructose 1,6-disphosphate, dihydroxyacetone phosphate or phosphate ion. The nature of the substrate C-1 phosphate binding site in this enzyme is discussed in the light of these and other results. (+info)
Effect of acrylamide on aldolase structure. I. Induction of intermediate states.
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Acrylamide is a fluorescence quencher frequently applied for analysis of protein fluorophores exposure with the silent assumption that it does not affect the native structure of protein. In this report, it is shown that quenching of tryptophan residues in aldolase is a time-dependent process. The Stern-Volmer constant increases from 1.32 to 2.01 M-1 during the first 100 s of incubation of aldolase with acrylamide. Two tryptophan residues/subunit are accessible to quenching after 100 s of aldolase interaction with acrylamide. Up to about 1.2 M acrylamide concentration enzyme inactivation is reversible. Independent analyses of the changes of enzyme activity, 1ANS fluorescence during its displacement from aldolase active-site, UV-difference spectra and near-UV CD spectra were carried out to monitor the transition of aldolase structure. From these measurements a stepwise transformation of aldolase molecules from native state (N) through intermediates: I1, T, I2, to denatured (D) state is concluded. The maxima of I1, T, I2 and D states populations occur at 0.2, 1.0, 2.0 and above 3.0 M of acrylamide concentration, respectively. Above 3.5 M, acrylamide aldolase molecules become irreversibly inactivated. (+info)
Effect of acrylamide on aldolase structure. II. Characterization of aldolase unfolding intermediates.
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Molecules of muscle aldolase A exposed to acrylamide change their conformation via I1, T, I2, D intermediates [1] and undergo a slow irreversible chemical modification of thiol groups. There is no direct correlation between activity loss and thiol groups modification. In the native enzyme two classes of Trp residues of 1. 8 ns and 4.9 ns fluorescence lifetime have been found. Acrylamide (0. 2-0.5 M) increases lifetime of longer-lived component, yet the transfer of aldolase molecules even from higher (1.0 M) perturbant concentration to a buffer, allows regain original Trp fluorescence lifetime. I1, detected at about 0.2 M acrylamide, represents low populated tetramers of preserved enzyme activity. T, of maximum population at about 0.7-1.0 M acrylamide, consists of meta-stable tetramers of partial enzymatic activity. These molecules are able to exchange their subunits with aldolase C in opposition to the native molecules. At transition point for I2 appearance (1.8 M acrylamide), aldolase becomes highly unstable: part of molecules dissociate into subunits which in the absence of perturbant are able to reassociate into active tetramers, the remaining part undergoes irreversible denaturation and aggregation. Some expansion of aldolase tetramers takes place prior to dissociation. D, observed above 3.0 M acrylamide, consists of irreversibly denatured enzyme molecules. (+info)
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.
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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. (+info)
Aldolase mediates the association of F-actin with the insulin-responsive glucose transporter GLUT4.
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To identify potential proteins interacting with the insulin-responsive glucose transporter (GLUT4), we generated fusion proteins of glutathione S-transferase (GST) and the final 30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of these carboxyl-terminal fusion proteins with adipocyte cell extracts revealed a specific interaction of GLUT4 with fructose 1, 6-bisphosphate aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in intact 3T3L1 adipocytes, which decreased following insulin stimulation. Introduction into permeabilized 3T3L1 adipocytes of fructose 1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents that disrupt the interaction between aldolase and actin, inhibited insulin-stimulated GLUT4 exocytosis without affecting GLUT4 endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These data suggest that aldolase functions as a scaffolding protein for GLUT4 and that glucose metabolism may provide a negative feedback signal for the regulation of glucose transport by insulin. (+info)
Serum samples of patients with rheumatoid arthritis contain a specific autoantibody to "denatured" aldolase A in the osteoblast-like cell line, MG-63.
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OBJECTIVE: To identify rheumatoid arthritis (RA) specific autoantibody and its antigen in the human osteoblast-like cell line, MG-63. METHODS: MG-63 cell extract was subjected to western blotting by using RA and normal serum samples as probes. The autoantigen was purified and its N-terminal sequence was determined by automated Edman degradation. The reactivity of denatured aldolase A was evaluated by immunoblotting. Screening by enzyme linked immunosorbent assay (ELISA) using the autoantibody was performed. RESULTS: 40 kDa protein was found only in the RA serum samples and it was identified as aldolase A. A polyclonal antibody for rabbit muscle aldolase A bound to the 40 kDa protein and reacted in preference with the denatured enzyme. Using ELISA for denatured rabbit aldolase A, the autoantibody was found in approximately 10% of RA patients, whereas it was not found in the other arthropathy and healthy adults. CONCLUSION: This 40 kDa anti-aldolase A autoantibody, which was identified only in serum samples of RA patients with severe bone erosion, could be related to a certain event that induces RA specific joint destructions. (+info)