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)

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

Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.
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PMID:Interactions between globular proteins and F-actin in isotonic saline solution. 165 57

A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep reaction using the exogenous or endogenous auxiliary enzymes aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. The highest activity was found in unfixed cryostat sections of rat liver when the incubation medium contained 17% (wt/vol) polyvinyl alcohol, 100 mmol/L Tris-maleate buffer (pH 8.4), 20 mmol/L fructose-6-phosphate, 2 mmol/L ATP, 2 mmol/L MgCl2, 5.9 mmol/L NAD+, 0.47 mmol/L 1-methoxyphenazine methosulfate, 5 mmol/L sodium azide and 5 mmol/L Nitro BT. The addition of auxiliary enzymes was not necessary to demonstrate maximum activity in rat liver. The specificity of the reaction was proven by the absence of any specific (test minus control) reaction when the incubation was performed in the presence of 25 mmol/L phosphoenolpyruvate, a competitive inhibitor of phosphofructokinase. Cytophotometric analysis revealed that linear relationships exist between the amount of specific reaction product formed and incubation time and the section thickness. The Km values for fructose-6-phosphate and the Vmax values were not significantly different in periportal and pericentral areas of livers from either normally fed or 24-hr-fasted rats. The homogeneous distribution of phosphofructokinase activity in the liver acinus is in line with biochemical findings using hepatocytes isolated from the two different areas showing that these cells contained similar amounts of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Homogeneous distribution of phosphofructokinase in the rat liver acinus: a quantitative histochemical study. 183 3

The affinity of baker's yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase was tested by using a fluorescence-probe technique with fluorescein isothiocyanate attached covalently to the enzymes. The dissociation constants of the enzyme-enzyme complexes, as well as the rate constants of association and dissociation, were determined. Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20 degrees C). The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species. Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non-hybrid complexes indicates that this ancient characteristic is probably conserved during evolution. A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metabolism.
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PMID:Interaction of enzymes involved in triosephosphate metabolism. Comparison of yeast and rabbit muscle cytoplasmic systems. 294 91

Srivastava and Bernhard [Srivastava, D. K. & Bernhard, S. A. (1986) Science 234, 1081-1086] have proposed that glycolytic enzymes form multienzyme complexes for the direct transfer of metabolites from the producing enzyme to the utilizing one. We have reinvestigated the evidence for direct transfer of NADH between its complexes with alpha-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27). The results reveal the following. (i) Proper treatment of the kinetics of and equilibrium data for the transfer of NADH between GPDH and LDH indicates that NADH transfer proceeds by a free-diffusion mechanism and not by direct transfer through a ternary complex. (ii) The koff for NADH from its GPDH complex is 60 sec-1 rather than 9.4 sec-1 in Tris.HCl buffer (pH 7.4) at 25 degrees C. With this value one can explain kcat = 50 sec-1 for LDH-catalyzed hydrogenation of pyruvate with GPDH-bound NADH as coenzyme. (iii) Steady-state kinetics show that LDH inhibits the GPDH-catalyzed reaction simply by reducing the concentration of free NADH. Similarly, aldolase inhibits the GPDH-catalyzed reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by binding to the substrate. The proposed direct transfer of NADH between GPDH and LDH is therefore mainly based on a misinterpretation of the experimental data.
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PMID:Reexamination of the kinetics of the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase and with lactate dehydrogenase. 319 95

Acute arterial occlusion of the extremities may result in severe and complex metabolic derangement. In order to evaluate the development and means of therapeutic control of metabolic derangements following the acute arterial occlusion of extremities, 32 adult mongrel dogs weighing between 7 and 15 kg underwent acute arterial occlusion by cross-clamping the infrarenal aorta. The experimental animals were divided into two groups: an untreated group, and treated group. The former was divided into three groups--12, 24 and 48 hour's arterial occlusive groups and the latter into two groups with 48 hour's arterial occlusion--tris (hydroxymethyl) aminomethane (THAM) and perfusion groups. Biochemical and electrolyte analyses were measured before occlusion, immediately before, and 1, 3, 12, 24 and 48 hours after the release of the occlusion. The SGOT, CPK, aldolase, creatinine and blood urea nitrogen levels rose after the release of the occlusion and were significantly higher in the 48 hour's group than in the 12 and 24 hour's occlusive groups. Among these enzymatic changes, the creatinine and urea nitrogen levels were high 48 hours after the release of the occlusion, though the others decreased with time after the occlusion release. The blood pH level fell after the occlusion in the untreated groups and these levels increased slowly after the release of the occlusion. However, there were no significant differences in the blood pH among the untreated groups. The acute arterial occlusion by cross-clamping the infra-renal aorta caused severe renal damage among the various organs. In the groups treated with THAM and perfusion, the SGOT, CPK, aldolase, creatinine and blood urea nitrogen levels remained almost at preocclusion levels after the release of the occlusion. There were statistically significant differences in these enzymatic changes between the treated group and the 48 hour's occlusive group without treatment. The blood pH levels in the treated groups showed minimal changes after the release of the occlusive, although there were no significant differences in the blood pH between the treated groups and the 48 hour's occlusive group without treatment. It was concluded that the intravenous administration of THAM and peripheral washing were effective against untoward metabolic changes occurring in the ischemic extremities.
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PMID:[Methods of suppression of a myonephropathic metabolic syndrome after infra-renal aortic occlusion]. 374 2

Acute arterial occlusion of the extremities may result in severe and complex metabolic derangements. In order to investigate therapeutic means of controlling such metabolic derangements, 19 adult mongrel dogs weighing between 7 and 15 kg underwent acute arterial occlusion by cross-clamping the infrarenal aorta. Clamping was released after 48 hours. The experimental animals were divided into three groups: an untreated group, a THAM group, and a perfusion group. Biochemical and electrolyte analyses were measured before and 1 hour after occlusion, and 1, 3, 12, 24, and 48 hours after the release of occlusion. The SGOT, creatinine, CPK, and aldolase levels rose after release of the occlusion and were significantly higher in the untreated group than in the THAM and perfusion groups. Among these enzymatic changes, the CPK level showed the largest increase. The serum potassium levels remained almost at preocclusion levels after release of the occlusion. It was concluded that the intravenous administration of THAM and peripheral washing were effective against untoward metabolic changes occurring in the ischemic extremities.
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PMID:Methods of suppression of myonephropathic metabolic syndrome. 403 Aug 79

A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0 degrees C, 0.15 M Tris.HCl buffer have been determined; a library of 913 amide-containing peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solutions, the use of this method for quantitative measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as molecular timers of biological events.
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PMID:Molecular clocks. 1115 75

Fructose 1,6-diphosphate (FDP) aldolase and 2-keto-3-deoxy-D-gluconate (KDG) aldolase the two key enzymes of Embden-Meyerhof-Parnas (EMP) and the nonphosphorolytic Entner-Doudoroff (ED) pathways respectively, were identified in cell-free extracts of four Aspergillus oryzae strains grown on D-glucose as sole source of carbon. A. oryzae NRRL 3435 gave the highest enzymatic activity for the two enzymes and selected for further studies. Studies on the properties of the two key enzymes indicated that the optimum conditions for the activities of FDP aldolase and KDG aldolases occurred at pH 8.5, 45 degrees C and pH 8.0, 55 degrees C, respectively. Tris-acetate buffer and phosphate buffer showed the highest enzymatic activity for these two enzymes respectively. KDG aldolase was stable at 55 degrees C for 60 minutes however FDP aldolase was found to be less stable above 45 degrees C. On the other hand the two aldolases showed a high degree of stability towards frequent freezing and thawing. Dialysis of the extracts caused a decrease in the enzymatic activity of KDG aldolase, and an increase in FDP aldolase activity. The addition of ethylene diamine tetraacetate to the crude extracts caused an inhibition of KDG aldolase, whileas FDP aldolase was not affected. Addition of MnCl(2), CoSO(4), MgCl(2) and ZnSO(4) to the dialyzed extracts increased the activity of KDG aldolase by 67%, 54%, 61% and 37%, respectively. On the other hand the addition of some metal salts caused an inhibition of FDP aldolase. The results obtained indicate the absence of evidence for the involvement of sulfhydryl groups in the catalytic sites of the two aldolases.
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PMID:Some properties of two aldolases in extracts of Aspergillus oryzae. 1567 61

HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.
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PMID:Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI. 1599 99


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