Biomedical subjects

K Eschrich

Publications and source records attributed to K Eschrich.

At least 55 records · Page 3Linked to original sources

PubMed1994

Determination of isozyme-specific mRNAs of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in glutamate obese rats by RNA probes.

To study the possible alterations in the metabolism of fructose 2,6-bisphosphate in glutamate induced obese rats, the mRNA levels of different isozymes coding for the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/F2,6Pase) were determined in various rat tissues obtained from fed and starved animals. For Northern analysis and dot blotting radioactively labeled RNA probes were synthesized by in vitro transcription. A modified ligase-free subcloning method was applied to obtain the plasmid vectors containing specific fragments of the cDNAs coding for the muscle, liver and heart isozymes of PFK-2/F2,6Pase. In liver but not in heart and skeletal muscle of glutamate obese rats the content of mRNA of PFK-2/F2,6Pase is significantly lower than in normal rats. As in normal rats short-term starvation has no effect on mRNA levels of the bifunctional enzyme. These results are compared with the maximum activities of PFK-2 and F2,6Pase determined in cell-free extracts prepared from the three organs.

Animals↗

PubMed1993

Role of Tyr201 and Tyr385 in substrate activation by p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The crystal structure of the enzyme-substrate complex of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4-hydroxybenzoate interacts with the side chain of Tyr201, which is in close contact with the side chain of Tyr385. The role of this hydrogen bonding network in substrate activation was studied by kinetic and spectral analysis of Tyr-->Phe mutant enzymes. The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr201-->Phe and Tyr385-->Phe) with the physiological substrate are comparable with those of the corresponding mutant proteins of p-hydroxybenzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Ballou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. Enzyme Tyr201-->Phe has a high Km for NADPH and produces only 5% of 3,4-dihydroxybenzoate/catalytic cycle. Unlike the wild-type enzyme, the Tyr201-->Phe mutant does not stabilize the phenolate form of 4-hydroxybenzoate. With enzyme Tyr385-->Phe, flavin reduction is rate-limiting and the turnover rate is only 2% of wild type. Despite rather efficient hydroxylation, and deviating from the description of the corresponding P. aeruginosa enzyme, mutant Tyr385-->Phe prefers the binding of the phenolic form of 4-hydroxybenzoate. Studies with substrate analogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4-fluorobenzoate differentially stimulates the stabilization of the 4 alpha-hydroperoxyflavin intermediate. Unlike wild type, both Tyr mutants produce 3,4,5-trihydroxybenzoate from 3,4-dihydroxybenzoate. The affinity of enzyme Tyr201-->Phe for the dianionic substrate 2,3,5,6-tetrafluoro-4-hydroxybenzoate is very low, probably because of repulsion of the substrate phenolate in a more nonpolar microenvironment. In contrast to data reported for p-hydroxybenzoate hydroxylase from P. aeruginosa, binding of the inhibitor 4-hydroxycinnamate to wild-type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including secondary binding of the inhibitor. Enzyme Tyr201-->Phe does not stabilize the phenolate form of the inhibitor. In enzyme Tyr385-->Phe, the phenolic pKa of bound 4-hydroxycinnamate is increased with respect to wild type. It is proposed that Tyr385-->Phe is involved in substrate activation by facilitating the deprotonation of Tyr201.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1992

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1991

Simple and rapid purification of F1-ATPase from bovine heart mitochondria by affinity chromatography.

A simple and rapid method for the isolation of bovine heart mitochondrial adenosine 5'-triphosphatase (F1-ATPase) was developed. Mitochondria were purified by differential centrifugation and stored frozen. After thawing. F1-ATPase was released by treatment with chloroform. Purification of the enzyme was achieved by polyethylene glycol precipitation followed by chromatography on Procion Navy H-ER beaded cellulose in the presence of MgCl2. F1-ATPase was eluted by ATP in the absence of MgCl2. The purity of the enzyme was proved by SDS-polyacrylamide-gel electrophoresis. The purified F1-ATPase showed slightly non-hyperbolic kinetics towards ATP and nearly complete inhibition in the presence of millimolar concentrations of ADP.

Adenosine Diphosphate↗

PubMed1991

Kinetics of 6-phosphofructo-1-kinase from a yeast mutant.

The steady state kinetics of 6-phosphofructo-1-kinase was determined in a cell-free extract obtained from a yeast mutant (DFY 250) and compared with the kinetic properties of the enzyme of a wild-type strain (DFY 1). 6-Phosphofructo-1-kinase from the DFY 250 strain shows a complex kinetic behaviour, which is qualitatively similar to, but quantitatively different from, that of normal yeast 6-phosphofructo-1-kinase. The mutant enzyme has a lower affinity to its activators fructose 6-phosphate, fructose 2,6-bisphosphate and AMP. The inhibiting effect of ATP on the mutant 6-phosphofructo-1-kinase is substantially weaker than on the wild-type enzyme. A complex interaction between fructose 6-phosphate and fructose 2,6-bisphosphate at the 6-phosphofructo-1-kinase from strain DFY 250 is reflected by a remarkable substrate inhibition by fructose 6-phosphate even at saturating fructose 2,6-bisphosphate. The kinetic data were fitted to different variants of the Monod-Wyman-Changeux model by nonlinear regression analysis. It turned out that the influence of fructose 6-phosphate, ATP, AMP and fructose 2,6-bisphosphate on the activity of 6-phosphofructo-1-kinase from wild-type and DFY 250 strain could be described by rate equations of essentially the same structure.

Adenosine Diphosphate↗

PubMed1990

Engineering of microheterogeneity-resistant p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

By site-directed mutagenesis, Cys-116 was converted to Ser-116 in p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens. In contrast to wild-type enzyme, the C116S mutant is no longer susceptible to oxidation by hydrogen peroxide and shows no reactivity towards 5,5'-dithiobis(2-nitrobenzoate). Crystals of the C116S mutant are isomorphous with the crystal form of wild-type enzyme. A difference electron density confirms the mutation made.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1990

A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract.

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate. In the reaction system the quasi-irreversible reactions catalyzed by 6-phosphofructo-1-kinase, pyruvate kinase, and fructose-1,6-bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose-1,6-bisphosphatase did not significantly influence the dynamic capabilities of the investigated reaction sequence. The kinetic properties of the cell-free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.

Adenosine Triphosphate↗

PubMed1990

Regulation of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by enzyme phosphorylation and sn-glycerol 3-phosphate.

The regulation of the Fru-6-P/Fru-2,6-P2 cycle by the cooperation of allosteric and covalent mechanisms was investigated in a reconstituted enzyme system under in vitro conditions. Phosphorylation of the bifunctional enzyme exerts a much stronger effect than sn-glycerol 3-phosphate in lowering the quasi-stationary concentration of fructose 2,6-bisphosphate and in increasing the critical concentration of the fructose phosphates, respectively. However, sn-glycerol 3-phosphate is able to strongly amplify the decrease of the quasi-stationary concentration of fructose 2,6-bisphosphate due to phosphorylation. The experiments can be described by a mathematical model involving rate equations for the dephosphorylated and the phosphorylated PFD-2 and FBPase-2. The results are compared with data from the literature obtained under in vivo conditions.

Animals↗

PubMed1988

Control of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by sn-glycerol 3-phosphate.

The kinetics of PFK-2 and FBPase-2 from rat liver were investigated with respect to the substrates and the effector sn-glycerol 3-phosphate. PFK-2 exhibits a hyperbolic response with respect to its substrates Fru 6-P and ATP. The inhibition of the activity of PFK-2 by sn-glycerol 3-phosphate could be described by assuming competition with Fru 6-P at the catalytic site. sn-Glycerol 3-phosphate activates the FBPase-2 and is capable of reversing partially the inhibition of the enzyme by Fru 6-P. The dynamics of the PFK-2/FBPase-2 cycle has been investigated in an enzyme system composed of PFK-2/FBPase-2, creatine kinase and creatine phosphate. sn-Glycerol 3-phosphate was found to decrease the quasi-stationary concentration of Fru 2,6-P2. The control of the PFK-2/FBPase-2 cycle by sn-glycerol 3-phosphate turned out most efficient at high concentrations of both sn-glycerol 3-phosphate and Fru 6-P. In addition, sn-glycerol 3-phosphate was found to increase the concentration control coefficient of Fru 2,6-P2 with respect to Fru 6-P.

Adenosine Triphosphate↗

PubMed1985

Influence of fructose 2,6-bisphosphate on the phosphofructokinase/fructose 1,6-bisphosphatase cycle.

In a reconstituted enzyme system multiple stationary states and oscillatory motions of the substrate cycle catalyzed by phosphofructokinase and fructose 1,6-bisphosphatase are significantly influenced by fructose 2,6-bisphosphate. Depending on the initial conditions, fructose 2,6-bisphosphate was found either to generate or to extinguish oscillatory motions between glycolytic and gluconeogenic states. In general, stable glycolytic modes are favored because of the efficient activation of phosphofructokinase by this effector. The complex effect of fructose 2,6-bisphosphate on the rate of substrate cycling correlates with its synergistic cooperation with AMP in the activation of phosphofructokinase and inhibition of fructose 1,6-bisphosphatase.

Fructose-Bisphosphatase↗

PubMed1985

Temporal organization of the phosphofructokinase/fructose-1,6-biphosphatase cycle.

The dynamic and functional organization of the fructose-6-phosphate/fructose-1,6-bisphosphate cycle has been investigated in an open and homogeneous reconstituted enzyme system containing phosphofructokinase, fructose-1,6-biphosphatase, pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerase. The properties of this system were analyzed by a model based on the kinetic properties of the individual enzymes. It could be shown that in a broad parameter region sustained oscillations arise. At low maximum activities of phosphofructokinase a domain of multiple stationary states occurs, in which stable stationary states can coexist with a stable oscillatory or with an alternate stable stationary state. The occurrence of oscillations and the emergence of alternate stationary motions are caused mainly by the reciprocal effect of the allosteric effectors AMP and fructose-2,6-bisphosphatase must be involved in the reaction network. The study of bisphosphatase. The attained states can either be glycolytic or gluconeogenic, their metabolic efficiencies depend mainly on the maximum activities of phosphofructokinase and fructose-1,6-bisphosphatase as well as on the supply of fructose-6-phosphate and fructose-1,6-bisphosphate. Efficient metabolic states arise only when both the enzyme concentrations and the rates of substrate supply favor either the glycolytic or the gluconeogenic mode of action. At medium maximum concentrations of the enzymes oscillations occur, in which glycolytic and gluconeogenic states are consecutively passed. A high rate of substrate cycling is observed only at the transitions between the functionally antagonistic phases of the periodicities. By this temporal organization the mean efficiency of the states is increased. The integration of fructose-2,6-bisphosphate as very sensitively acting activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase gives rise either to emergence of oscillations or of their extinction. Generally, the glycolytic mode is favored by this effector because of its stimulatory action on the phosphofructokinase activity.

Fructose-Bisphosphatase↗

PubMed1985

Glycolytic and gluconeogenic states in an enzyme system reconstituted from phosphofructokinase and fructose 1,6-bisphosphatase.

Transitions between glycolytic and gluconeogenic states have been investigated in an open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerase. The direction of substrate flow was found to depend on the maximum activities of phosphofructokinase and fructose 1,6-bisphosphatase as well as on the influx concentrations of fructose 6-phosphate and fructose 1,6-bisphosphate. At high and low maximum activities of phosphofructokinase and fructose 1,6-bisphosphatase unique and stable stationary states occur, whereas at intermediate enzyme concentrations sustained oscillations emerge. Stationary states with a low rate of substrate cycling demand both appropriate enzyme concentrations and an adequate substrate supply. Accordingly, transitions between efficient glycolytic and gluconeogenic states require changes of the enzyme concentrations and of the supply of substrates. Such transitions exhibit a transient oscillatory response. The sustained oscillations generated at intermediate activities of phosphofructokinase and fructose 1,6-bisphosphatase lead to a significant diminution of the rate of substrate cycling when compared with the respective steady state values. During the oscillations glycolytic and gluconeogenic states are consecutively passed through. Because of this a temporal organization of the antagonistic reactions is achieved. In our system the kinetic organization of the two opposite reactions is mainly brought about by the reciprocal allosteric effects of AMP on the activities of the two enzymes.

Adenosine Triphosphate↗

PubMed1985

Dynamic structures of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in a reconstituted enzyme system.

The dynamics of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle was investigated in an open and homogeneous system reconstituted from purified enzymes. In addition to phosphofructokinase and fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerases are involved. The time evolution of the metabolite concentrations is governed by a set of differential equations which take into account flow processes and enzymic conversions of metabolites. Depending on the experimental parameters stable attractors, multiple states and sustained oscillations occur. The main source of the nonlinear dynamics is the reciprocal effect of AMP on the activities of phosphofructokinase and fructose 1,6-bisphosphatase. States are characterized by the net flow rates of substrates and by the rate of futile substrate cycling. For efficient glycolytic states high ratios between the influx rates of fructose 6-phosphate and fructose 1,6-bisphosphate and between the maximum activities of phosphofructokinase and fructose 1,6-bisphosphatase must be maintained, while for an efficient gluconeogenic mode the reverse must hold. Fructose 2,6-bisphosphate exerts reciprocal effects on the activities of phosphofructokinase and fructose 1,6-bisphosphatase. In dependence on the experimental conditions fructose 2,6-bisphosphate was found either to generate or to extinguish oscillations.

Animals↗

PubMed1984

Effect of enzyme concentrations on sustained oscillations in the fructose 6-phosphate/fructose 1,6-bisphosphate-cycle.

Sustained oscillations were investigated in an open and homogeneous enzyme system reconstituted from phosphofructokinase, fructose 1,6- biphosphatase , pyruvate kinase, adenylate kinase, and glucose 6-phosphate isomerase. The generation of oscillations if mainly due to antagonistic kinetic effects of AMP on phosphofructokinase and fructose 1,6- biphosphatase under the experimental conditions applied. Sustained oscillations were obtained in a broad range of maximum activities of phosphofructokinase or fructose 1,6- biphosphatase are increased. At low maximum activities of the two enzymes oscillations arise, which form a folded limit cycle, while by increasing concentrations of phosphofructokinase or fructose 1,6-bisphosphatase sustained oscillations are introduced, which are restricted to two dimensions.

Adenylate Kinase↗

PubMed1983

Sustained oscillations in a reconstituted enzyme system containing phosphofructokinase and fructose 1,6-bisphosphatase.

In a reconstituted open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase sustained oscillations could experimentally be generated. The approach is based on a stirred flow-through reaction chamber. The periodic motions of the reactants are mainly caused by the antagonistic allosteric effects of the adenine nucleotides on the activities of the phosphofructokinase and fructose 1,6-bisphosphatase.

Allosteric Regulation↗

PubMed1983

Diminution of stationary enzyme activities at increases of pyruvate kinase concentration in a reconstituted enzyme system.

In a homogeneous and open enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose 6-phosphate isomerase the consequences of variations of the enzyme concentrations on the stationary enzyme activities have been investigated. An unexpected behavior was observed upon variation of the maximum activity of pyruvate kinase. Depending on the experimental conditions an increase of the concentration of pyruvate kinase resulted either in a diminution or in a stimulation of the stationary activity of this enzyme. An increase of the maximum activity of phosphofructokinase, however, stimulates both the activities of phosphofructokinase and pyruvate kinase. The experimental results are interpreted in terms of a mathematical model, based on the kinetic properties of the enzymes involved. The correlation between the observed changes of the activities of phosphofructokinase and pyruvate kinase and the appearance of multiple stationary states is discussed.

Adenylate Kinase↗

PubMed1983

Oscillations in the phosphofructokinase-fructose 1,6-bisphosphatase cycle. I. Purification and kinetic characterization of fructose 1,6-bisphosphatase from pig liver.

A rapid and effective purification procedure for pig liver fructose 1,6-bisphosphatase in neutral form is described. The procedure involves heat treatment and chromatography with CM-Sephadex with specific elution of the enzyme by fructose 1,6-bisphosphate and AMP. The enzyme was found suitable for integration into a reconstituted enzyme system in which the generation of oscillations is investigated. The kinetic properties of fructose 1,6-bisphosphatase are studied under conditions compatible to those applied for the investigation of the dynamic behaviour of the reconstituted system (pH 6.6, presence of inorganic phosphate). The enzyme is significantly inhibited by AMP and fructose 6-phosphate. The substrate fructose 1,6-bis phosphate has a high affinity to the enzyme and was found weakly inhibiting even at high concentrations. The kinetic results are interpreted in terms of a mathematical model which reflects the interaction of the various effectors with the enzyme.

Animals↗

PubMed1983

Oscillations in the phosphofructokinase--fructose 1,6-bisphosphatase cycle. II. Influence of fructose 1,6-bisphosphatase on the character of oscillatory states.

The dynamic behaviour of an open futile cycle composed of phosphofructokinase and fructose 1,6-bisphosphatase has been investigated in a homogeneous reconstituted enzyme system in which the two enzymes cooperate with pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerase. By the kinetic cooperation of phosphofructokinase and fructose 1,6-bisphosphatase various types of dynamic patterns can be generated. In the absence of fructose 1,6-bisphosphatase oscillations do not occur. As experimentally demonstrated, in a definite region of maximum activities of fructose 1,6-bisphosphatase sustained oscillations originate while at higher concentrations of the enzyme damped oscillations appear. At increasing concentrations of fructose 1,6-bisphosphatase damping is strengthened. Finally, at very high concentrations of fructose 1,6-bisphosphatase the metabolites relax monotonously to the stationary states. The observed oscillatory phenomena are mainly caused by the antagonistic effects of AMP on the kinetics of phosphofructokinase and fructose 1,6-bisphosphatase.

Adenylate Kinase↗