[A titrographic method for determining the glycolysis flow rate with Plasmodium berghei-infected red blood cells]. / Ein titrographisches Verfahren zur Bestimmung der Glykolyse-Flussrate mit Plasmodium berghei infizierter roter Blutzellen.

A procedure is described for the elucidation of the glycolytic flux rate of red blood cells infected with the malarial parasite Plasmodium berghei. It is based on the titration of the protons originating from the glycolytic lactate accumulation. Compared with traditional methods of biochemical measurements of glucose consumption or accumulation of lactate the proposed procedure shows the following advantages: continuously measurement is possible; constancy of the pH-value during the measurement; lower amounts of biological material are necessary (increased sensitivity) increased accuracy; The method can also be applied to studies on other cells and species with normal or increased glycolytic flux rate.

Phosphofructokinase from Plasmodium berghei: a kinetic model of allosteric regulation.

As in mammalian cells, phosphofructokinase (PFK) is of major regulatory importance in the glucose metabolism of Plasmodium berghei. The malarial enzyme shows allosteric properties similar to PFK from various sources; it is activated by fructose-6-phosphate and inhibited by ATP, but differs with respect to allosteric regulation. Enzyme activity is only marginally increased by AMP, a potent activator of many phosphofructokinases. Phosphoenolpyruvate, which is reported to inhibit PFK activity, efficiency activates the malarial enzyme. No activation by ADP was observed. Instead, ADP inhibits the enzyme non-allosterically and competitively to the substrate MgATP. Phosphate stimulates the catalytic activity of malarial PFK independently of the activation by F6P and PEP.

Glucose-6-phosphate dehydrogenase from Plasmodium berghei: kinetic and electrophoretic characterization.

Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase (G6PD) in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. From infected rat erythrocytes the parasites were isolated, washed, and lysed. G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B, although the separation of the malaria-specific enzyme from that of the host cell was not complete. Malarial G6PD significantly differed from the red cell enzyme with respect to its electrophoretic properties. In cellulose acetate electrophoresis, a band with catodic mobility was observed in addition to the anodically mobile host cell enzyme at pH 7.0. The subunits of the parasite-specific G6PD have a molecular weight of 55 kDa in contrast to 59 kDa of red cell G6PD subunits. The enzyme from P. berghei shows no cross-reactivity with polyclonal antibodies against G6PD from rat erythrocytes. Thus, a close evolutionary relationship between both proteins and the presence of proteolytic modifications could be excluded. The Km value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.

Phosphofructokinase from Plasmodium berghei. Influence of Mg2+, ATP and Mg2(+)-complexed ATP.

The control enzyme phosphofructokinase is of regulatory significance in the metabolism of glucose by the malarial parasite Plasmodium berghei. (1) The enzyme was partially purified from erythrocytic stages of P. berghei by precipitation with poly(ethylene glycol) and chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. (2) Similarly to various other phosphofructokinases, the enzyme from P. berghei shows an allosteric behaviour. It is activated by fructose 6-phosphate and inhibited by ATP. (3) The effects of Mg2(+)-complexed ATP, free ATP and Mg2+ were studied by keeping constant the concentration of one of these and varying the concentrations of the other two. (4) The enzyme is shown to be allosterically inhibited by free ATP and by higher concentrations of Mg2+. Compared with phosphofructokinase of erythrocytes, inhibition by ATP is weaker by two orders of magnitude. Mg2(+)-complexed ATP has no effect on allosteric regulation. (5) The proposed kinetic model provides an adequate description of the data.

Classification of glucose-6-phosphate dehydrogenase variants by multivariate statistical analysis.

Using principal component analysis a two-dimensional presentation of kinetic parameters from various G6PD-variants was obtained from which similarities between individual enzymes became detectable. Conclusions could be drawn as to how far the kinetic parameters contribute to the discrimination between different variants. An objective classification of G6PD-variants was achieved by application of cluster analysis. The proposed methods provide an effective means for differential elucidation of G6PD-variants and other heterogeneous enzymopathies.

Glucose-6-phosphate dehydrogenase from Plasmodium berghei: kinetic and electrophoretic characterization.

Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. After separating the parasites from infected RBC the G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B. In cellulose acetate electrophoresis malarial G6PD significantly differs from the red cell enzyme. The subunits of the parasite-specific G6PD have a molecular weight of 55 kD in contrast to 59 kD of the RBC enzyme. G6PD from P. berghei shows no cross-reactivity with antibodies against G6PD from rat erythrocytes. The Km-value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.

[Classification of glucose-6-phosphate dehydrogenase enzymopathies: evaluation of kinetic parameters using multivariate methods]. / Klassifizierung von Glucose-6-Phosphatdehydrogenase-Enzymopathien: Auswertung kinetischer Parameter mittels multivariater Methoden.

The dependence between 9 kinetic parameters of glucose-6-phosphate dehydrogenase from 13 normal controls and 78 G6PD-deficient patients from the GDR and other socialist countries has been investigated by multivariate statistical methods. Using principal component analysis a two-dimensional presentation of the data was obtained from which similarities between individual enzyme variants became detectable. Conclusions could be drawn in how as far the kinetic parameters contribute to the discrimination between different variants. An objective classification of G6PD-variants was achieved by application of cluster analysis. The proposed methods provide an effective means for differential elucidation of G6PD-enzymopathies and should be also useful in the case of other enzymopathies.

Regulation of the energy metabolism of Plasmodium berghei.

Energy metabolism of malaria parasites was investigated in P. berghei infected red blood cells of rat. Although Plasmodia contain mitochondria most of their ATP is formed by glycolysis. Lactate formation is two orders of magnitude higher than in noninfected erythrocytes. The coupling of respiration and glycolysis is very loose, a Pasteur-effect was not found. The key enzymes of glycolysis hexokinase and phosphofructokinase have been partially purified and kinetically characterized. The kinetic properties of both enzymes significantly differ from those of erythrocytes. They are less efficiently inhibited and PFK is activated only by PEP, Fru6P and Pi. The high rate of glycolytic proton formation in Plasmodia inhibits the PFK and thus the anaerobic energy metabolism of the host cell but not that of the parasite. Nevertheless the ATP concentrations in the host and the parasite compartment were found to be nearly identical. This supports the assumption that the parasites make ATP available to their host cell, probably by an adenine nucleotide translocator.

Mathematical modelling of metabolic pathways affected by an enzyme deficiency.

The regulation of metabolic pathways of the red cell affected by an enzyme deficiency is studied on the basis of comprehensive mathematical models. The main steps of such a theoretical approach are outlined considering individual alterations in the kinetic properties of two regulatory enzymes: pyruvate kinase and glucose-6-phosphate dehydrogenase. It is demonstrated that mathematical modelling helps to relate the observed changes of cellular quantities as shortened life-span, or as resistance against oxidative stress to alterations in the metabolic regulation.

Estimating the degree of infection of Plasmodium berghei infected red blood cells by evaluation of pyruvate kinase activity.

A procedure is proposed to estimate the parasitemia of red blood cells infected with P. berghei by determination of pyruvate kinase activity. The activity of this enzyme shows a linear dependence not on the relative number of infected cells but on the average number of parasites per red blood cell. To provide a transformation between these two parameters a theoretical model of multiple infections of blood cells is derived, the accuracy of which was proved experimentally.

A kinetic model of phosphofructokinase from Plasmodium berghei. Influence of ATP and fructose-6-phosphate.

Phosphofructokinase (PFK) from the malarial parasite Plasmodium berghei shows the following kinetic features: the more the pH is decreased, the more the enzyme is inhibited by ATP; in contrast to PFK from erythrocytes, this inhibition is less potent by two orders of magnitude; as in the red cell, fructose-6-phosphate (F6P) is a positive effector. Kinetic modelling of PFK from P. berghei has been performed by taking the pH-dependence of activity into regard, implicitly by the estimation of pH-dependent kinetic parameters for the inhibition by ATP and the activation by F6P and explicitly by the assumption of protonation-steps involved in allosteric regulation. By means of a novel procedure of model discrimination [D. Buckwitz and H.-G. Holzhütter: A new method to discriminate between enzyme-kinetic models. In: Application of Computational Methods in Medicine (Györi, I., ed.), Akademai, Budapest, in press] we have selected among several kinetic models the best rate equation which provides an adequate quantitative description of the kinetic behaviour of the enzyme in the relevant ranges of substrate concentrations and pH (5.8-7.6). It thus becomes clear how the highly increased glycolytic flux in malaria-infected cells could be affected through PFK.

Heterogeneity of glucose-6-phosphate dehydrogenase enzymopathies in the GDR.

G6PD variants of 13 patients from 12 German families with different clinical symptoms have been characterized kinetically. Vmax G6PD was nearly zero in red blood cells of all carriers. Therefore G6PD variants were isolated from leucocytes, which proved to be a suitable source for analysis of instable G6PD variants. The testing program included KmG6P, KmNADP, Ki values of NADPH, ATP and 2,3 P2G, rate of utilization of dG6P, Gal6P, dNADP, NAD, and pH dependence. From the results obtained one can conclude that all analyzed G6PD variants represent individual mutations. The degree of metabolic dysregulation can be explained by the different kinetic and physico-chemical properties of these G6PD variants.

Kinetic model of glucose-6-phosphate dehydrogenase from red blood cells. Parameter estimation from progress curves and simulation of regulatory properties.

A kinetic model of human and mouse glucose-6-phosphate dehydrogenase is presented which takes into account the substrates and all inhibitors of significant importance in the red cell. The parameter values were estimated by analysis of progress curves. The applicability of a new method based on non-linear regression to complex enzyme kinetics was proved. The in vivo-regulation of glucose-6-phosphate dehydrogenase is examined by determining elasticity coefficients and by using simple simulation experiments. The model is convenient to describe the behaviour of enzyme activity under physiological conditions.