Transient experimental anemia in cholesterol-fed rabbits induces systemic overexpression of the reticulocyte-type 15-lipoxygenase and protects from aortic lipid deposition.

Oxidative modification of low-density lipoprotein has been implicated in atherogenesis and the lipid peroxidizing enzyme 12/15-lipoxygenase (12/15-LOX) was suggested to be involved. For this study, we induced a strong and long-lasting systemic overexpression of the 15-LOX, in female New Zealand White rabbits by transient experimental anemia. After the hematopoietic parameters had returned to normal, these animals and age-matched controls were fed a lipid-rich Western-type diet for 10 weeks. Analyzing the lipid deposition in the aortic wall, we found that the 15-LOX overexpressing rabbits deposited significantly (P<0.01) less cholesteryl linoleate in the thoracic aorta than the corresponding controls. Similar results were obtained when free cholesterol and cholesteryl oleate were quantified. However, in the aortic arch where lipid deposition was much more severe a similar trend was observed, but the effects were not significant any more. Comparative determination (lipoxygenase overexpressing vs. control animals) of various plasma parameters as well as histological inspections of major organs did not reveal any indications for major organ malfunction. These data suggest that transient experimental anemia, which is accompanied by a long-lasting overexpression of the reticulocyte-type 15-LOX protects cholesterol-fed rabbits from lipid deposition in the aortic wall.

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.

Proliferating alveolar macrophages in BAL and lung function changes in interstitial lung disease.

In interstitial lung disease, the number of alveolar macrophages (AMs) can be increased. This may be caused by recruitment of precursor cells from peripheral blood and/or local proliferation in the lung. We therefore analysed proliferation, by studying both the expression of the nuclear proliferation antigen, Ki67, and the deoxyribonucleic acid (DNA) content, using the Feulgen reaction followed by cytometry. The patients had interstitial lung disease, i.e. sarcoidosis (n = 20), extrinsic allergic alveolitis (n = 20), idiopathic lung fibrosis or lung involvement in collagen-vascular disease (n = 19). In all patient groups there was a significant increase in proliferating AMs compared to healthy controls (4.2 versus 1.4% Feulgen, 2.1 versus 0.5% Ki67), with a significant correlation between these two parameters. A positive correlation was also found in bronchoalveolar lavage (BAL) between numbers of lymphocytes and proliferating cells in sarcoidosis and in fibrosis. In fibrosis, numbers of eosinophils and proliferating cells were also positively correlated. Our main finding was, however, a positive correlation between numbers of proliferating cells (Feulgen) and lung function parameters, especially vital capacity and oxygen tension (PO2) at rest, in patients with sarcoidosis and lung fibrosis. By contrast, in extrinsic allergic alveolitis, no correlation could be observed between proliferating cells and cell population or lung function. Our results suggest that local proliferation of macrophages is an important element in interstitial lung disease.

[Diagnosis of genetically determined diseases]. / Diagnostik genetisch bedingter Erkrankungen.

Enzymopathies of pyruvate kinase are caused by defects of structural genes forming stable and unstable mutants, respectively. Stable mutants of PK are characterized by high S0,5 PEP and nearly unchanged Vmax. A decrease of PEP affinity can be the result from a very high allosteric constant L0 or from an increased KPEP. From the pattern of PAGE can be concluded that stable PK mutants are tetraheteromers composed of two normal and two shortened polypeptide chains. We suppose that this is the result of a mutation of a codon which stops the polypeptide synthesis of PK earlier. Most PK mutants are unstable. They are characterized by low catalytic activity and high PEP affinity. The kinetic properties of unstable mutants are changed posttranslational by proteolytic modifications. Furthermore low S0,5 PEP values result from a persistence of the isoenzyme PK-K in reticulocytes and erythrocytes, respectively. A prenatal diagnosis of PK enzymopathies can be carried out with a very small blood volume by using the method of isoelectrophoretic focussing.

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.

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.

Nucleotide status in erythrocytes of rats infected with Pl. berghei.

Nucleotide concentrations in erythrocytes of rats infected with Plasmodium berghei were measured by ion-pair reversed-phase HPLC. UTP and GTP levels were higher in highly infected red blood cells obtained after density separation. The infected red blood cells possess higher hypoxanthine, adenine, and adenosine levels.

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.

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.

[Energy metabolism of erythrocytes in pyruvate kinase enzymopathies]. / Energiestoffwechsel roter Blutzellen bei Pyruvatkinase-Knzymopathien.

Until now pyruvate kinase enzymopathies have been described only for red blood cells. On the basis of these results special structural properties of the erythrocyte PK was assumed, which are not yet totally established. PK defects may cause a nonspherocytic hemolytic anemia. This enzymopathy is characterized by a polymorphism, which is expressed in more than 5 different pathological variants. Up to now 16 cases of PK deficiency have ben diagnozed in the GDR. The following parameters are used for the characterization of the PK: the PEP-dependance, the inhibition by ATP and alanine, the specificity to nucleotides, the stability to temperature and urea and the maturation dependence. Two pathological variants of the PK with a decreased PEP-affinity are described. Furthermore the differences in the energy metabolism of the red blood cells of these two patients under aerobic and anaerobic conditions are discussed.

[Control of glycolysis in magnesium deficiency: studies on intact red cells and hemolysates]. / Kontrolle der Glykolyse bei magnesiummangel: Untersuchungen an intakten roten Blutzellen und Hämolysaten.

The behaviour of glycolytic flux and glycolytic metabolic concentrations was studied under conditions of magnesium deficiency. The Mg-deficiency was produced in whole animals (rats) by feeding a diet almost completely free of Mg and in hemolysates of men by the addition of a chelating agent. The results show that the decrease of the free Mg-level is diminished by partial destruction of ATP and 2,3-DPG. The analysis of the control strength of the overall flux leads to the conclusion that the decrease of the glycolytic rate is caused by an inhibition of the hexokinase-phosphofructokinase-control system. The decrease of the MgATP-Complex and free Mg++-level explains the diminished phosphorylation of glucose by the hexokinase. The ATP-inhibition of the phosphofructokinase is amplified by a small increase of free ATP-concentration and a simultaneous decrease of the Fru-6P-level. The increase of the PEP-level is caused by the diminished free Mg++ and MgATP-complex and does not demonstrate a larger control strength of the pyruvate kinase.