Amino-acid composition of pyruvate kinase M2 isoenzyme variants from rat liver and Morris hepatoma 7777.

Cytosolic fractions B (salted out between 51-70% ammonium sulphate saturation) from rat liver and Morris hepatoma 7777, containing pyruvate kinase (EC 2.7.1.40) M2 isoenzymes, were purified by affinity chromatography on Blue Sepharose CL-6B. When compared by polyacrylamide gel electrophoresis at pH 8.3, all three M2 pyruvate kinase variants from Morris hepatoma 7777 had lower mobilities (alpha2, beta2, gamma3) than the three corresponding variants (alpha1, beta1, gamma2) from normal rat liver. Using an automatic amino-acid analyser, significant differences in selected amino-acid content have been found in corresponding highly purified gamma3 and gamma2 variants from Morris hepatoma and normal rat liver, respectively. The gamma3-variant of the Morris hepatoma M2 isoenzyme had twice the amount of L-tyrosine and L-cysteine, and a content of L-serine higher by 20% than the corresponding gamma2 variant of the normal rat liver M2 isoenzyme. It contained, however, significantly less dicarboxylic amino acids which explains its lower electrophoretic mobility. It showed also a decrease (by about 10%) in several other amino-acid content, corresponding to a 10% decrease in the tumour enzyme molecular mass.

Changes in ATP level and iron-induced ultra-weak photon emission in bull spermatozoa, caused by membrane peroxidation during thermal stress.

ATP level, cell motility and viability, oxygen uptake, pyruvate kinase activity, and ultra-weak photon emission (UPE) induced by red-ox Fe(2+)-ascorbate cycling system were studied in fresh, in previously equilibrated in a glycerol diluent, and in cryopreserved bull spermatozoa, exposed to thermal stress by incubation of the cells at 44 degrees C. A sharp drop in motility and viability of fresh spermatozoa and even more so, of equilibrated and cryopreserved cells was accompanied by accumulation of ATP. When cell movement was totally inhibited, ATP utilization was decreased, while chemical energy continued to be produced by cell pyruvate kinase, one of the key glycolytic enzymes, which in spermatozoa is very active (6500 IU/g protein) and insensitive to feed-back inhibition by excess of ATP and L-cysteine. Accumulation of ATP during incubation at 44 degrees C in 0.9% NaCl was accompanied by rapid decrease in oxygen consumption by fresh spermatozoa and an increase in Fe(2+)-ascorbate induced UPE, followed by a sharp decrease in ATP level observed at the end of induced UPE measurement. The increase in photon emission due to lipid peroxidation was highly correlated with the increase in cell ATP level caused by thermal stress.

Tumor-specific pyruvate kinase isoenzyme M2 involved in biochemical strategy of energy generation in neoplastic cells.

The differences in properties of pyruvate kinase (EC 2.1.7.40) from normal tissues and animal or human tumors are described and their significance for various metabolic abnormalities is reviewed. The tumor variant gamma3 from M2 isoenzyme of pyruvate kinase sensitive to L-cysteine inhibition, when over-expressed, can be used as a marker of tumorigenic transformation. It seems that this variant represents a tumor-specific oncoprotein, involved in a novel metabolic strategy of energy generation during increased cell proliferation.

N-acetylneuraminic acid, phosphate and thiol groups of pyruvate kinase isoenzymes from Morris hepatoma 7777 and normal rat liver.

The highest amount of N-acetylneuraminic acid (AcNeu) was found in pyruvate kinase isoenzyme L from normal rat liver (24 moles/mole of enzyme tetramer), with the highest electrophoretic mobility. On the other hand, isoenzyme M2 from Morris hepatoma 7777, with the lowest electrophoretic mobility, had the lowest AcNeu content (5 moles/mole of enzyme tetramer). This tumour isoenzyme M2 of pyruvate kinase was, however, characterised by the highest phosphate content (12 moles/mole protein), in comparison to isoenzyme L (3 moles/mole protein) or normal liver isoenzyme M2 (6 moles/mole protein). This could indicate a regulatory change caused by reversible enzyme phosphorylation and dephosphorylation or sialization and desialization. Despite these differences, the sum of the two negatively charged residues was lower in tumour pyruvate kinase isoenzyme M2, with the slowest migration rate, than in normal rat liver isoenzyme M2. Moreover, isoenzyme M2 from tumour material, in comparison with isoenzyme M2 from normal rat liver, had a twice as high content of thiol groups (20 moles/mole protein), especially of free and superficially located ones, than the isoenzyme M2 from normal liver (10 moles/mole protein). This may explain abnormal susceptibility of tumour isoenzyme M2 to stereospecific inhibition by exogenous L-cysteine, and indicate genetically dependent changes in amino-acid content of tumour enzyme which take place during cell tumourigenic transformation.

In vitro inhibitory effect of chitosan and its degradation products on energy metabolism in Ehrlich ascites tumour cells (EAT).

Chitosan, a product of depolymerization and deacetylation of chitin, as polycation, diminishes glycolysis, i.e. aerobic lactate formation both, in Ehrlich ascites tumour (EAT) intact cells and in their cytosolic fraction. By inhibiting glycolysis, chitosan decreases also glucose uptake and ATP level in intact cells. Chitosan does not exert a similar inhibitory effect on glycolytic activity of mouse normal liver and muscle supernatants. It has been found that a decrease in glycolytic activity and cell ATP level in tumour cells is caused by inhibition of tumour specific variant of pyruvate kinase from fraction B (isoenzyme M2).

The effect of lead on lactate formation, ATP level and membrane ATPase activities in human erythrocytes in vitro.

Lead ions inhibit aerobic glycolysis and diminish ATP level in human erythrocytes in vitro. Magnesium partly abolishes this inhibitory effect of lead on lactate formation by stimulation of Mg-dependent enzymes. Lead ions also inhibit the (Na, K) ATPase activity of the erythrocyte membranes. This effect is seen after the direct adding of lead acetate to erythrocyte ghosts, as well as in the ghosts obtained after preincubation of intact erythrocytes with lead acetate, prior to cell membrane isolation (Ca, Mg) ATPase is less sensitive to lead and (Mg) ATPase is practically insensitive. During these studies a protective effect of glucose was found. The inhibition of (Na, K) ATPase by lead ions, observed after incubation of human erythrocytes, as an experimental model, may indicate a similar sensitivity of membrane (Na, K) ATPases from other cells during their in vitro exposure to lead, especially from the nervous cells, where (Na, K) ATPase participates in the process of cell surface repolarization.

Electrophoretic pattern of cytosolic pyruvate kinase fractions A and B (type L and M2) from normal rat liver and Morris hepatoma 7777.

Cytosolic pyruvate kinase fractions A and B obtained by salting out procedure from normal rat liver and Morris hepatoma 7777, purified by affinity chromatography on Blue Sepharose CL-6B, have shown similar electrophoretic patterns in polyacrylamide gel at pH 8.3 to previously studied pyruvate kinase extracts from chromatin of cell nuclei. Three variants (alpha 1, beta 1, gamma 1) from normal rat liver pyruvate kinase fraction A (type L) had the greatest electrophoretic mobility, showed sigmoidal kinetics in relation to 2-phosphoenolpyruvate (PEP), and sensitivity to ATP and fructose 1,6-diphosphate (FDP). The fraction A dominated over normal liver fraction B (type M2), which in electrophoresis showed a slower gamma 2 variant, similar to the fraction A of hepatoma. All variants from fractions B of normal liver and A of hepatoma had linear kinetics and were sensitive to ATP but not to FDP. The greatest differences showed pyruvate kinase fraction B from Morris hepatoma. Its all variants alpha 2, beta 2, gamma 3 were cathodic and had linear kinetics in relation to PEP. They all were insensitive to normal signal molecules (ATP and FDP). The gamma 3 alkaline variant acquired sensitivity to inhibition by L-cysteine. Showing several-fold higher activity, much greater affinity to the main substrate, and a lack of sensitivity to feed-back inhibition by ATP, it was responsible for a high rate of aerobic glycolysis and diminution of the Pasteur effect in metabolic studies. It was probably encoded during oncogene activation and plays a special role in different metabolic strategies of tumour cells.

Effect of butobendin on dopamine beta-hydroxylase in vitro.

It has been found that dopamine beta-hydroxylase (DBH) (E.C.1.14.17.1), a key enzyme in the multistep process of adrenaline formation, isolated from blood serum by fractionated salting out procedure, is an allosteric enzyme with a sigmoidal kinetics and positive cooperativity in binding of the substrate and effectors. Butobendin, a double ester of 2-aminobutanol and trimethoxybenzoic acid, a new interesting compound showing various pharmacological and metabolic properties, inhibits DBH activity stereospecifically, only in the configuration 2S,2'S, decreasing DBH Vmax. This noncompetitive inhibitory effect of butobendin was stronger than the feed-back DBH inhibition by adrenaline and noradrenaline or by quinidine used as a standard antiarrhythmic compound. At high butobendin concentration (1.55 microM) the DBH activity was stabilized at about 50% of initial activity. This indicates that tetrameric enzyme binds substrate to half-of-its-binding-sites. At low substrate (tyramine) concentration (1-5 mM), a sigmoidal kinetics was preserved only at low butobendin concentration (0.155-0.31 microM). At higher butobendin concentrations (0.77 microM) a linear kinetics of DBH appeared and Km value decreased, indicating an increase in affinity of enzyme to the substrate. Interesting regulatory properties of DBH and butobendin action deserve further attention, especially for understanding a therapeutic action of butobendin in some types of heart rhythm disturbances.

Reduced levels of alpha-1-antitrypsin in children exposed to high levels of air pollution.

Health risks associated with environmental pollution, particularly in Eastern Europe, are a cause for concern. In order to gain some insight into the effects of exposure to high levels of air pollution particularly by heavy metals, we measured the concentration of nine plasma proteins in plasma samples from newborn babies and 5- to 6-year old children living in heavily polluted areas of Poland, as well as from some newborns and adults from unpolluted areas of Poland and control samples within the local Southampton population. The concentrations of all proteins measured were in or close to their expected range, with the exception of alpha 1-antitrypsin (alpha 1AT). The concentration of this protein was lower in both groups of Polish children from the polluted areas, particularly in the 5- to 6-year old group. These children were shown to be phenotypically normal. Since adult levels of alpha 1AT are usually reached within 2 weeks postpartum, these findings raise the possibility that continued exposure to high levels of pollution may lead to reduced levels of alpha 1AT in plasma; this may compound the impairment of lung function in children living in highly polluted areas.

Environmental problems of Cracow and their health consequences.

In 1983, 27 areas of ecological hazard were recognized in Poland. The situation was especially bad in the south of Poland, in the region of Katowice and Cracow. In this paper the synergistic effects of various air pollutants on the residents' health were analysed. Results show that, besides the threat to future generations, pollution reduces somatic strength and mental ability by diminishing biological energy formation.

Effect of environmental fluorides on key biochemical processes in humans.

Several biochemical parameters connected with energy metabolism were studied in the inhabitants living in the neighbourhood of the Aluminium Smelter and in inhabitants of Chorzów, one of the most polluted towns in the Upper Silesian Industrial Region, chronically exposed to environmental fluorides and other toxic compounds. In people exposed to environmental fluorides excessive excretion of fluorides in urine was noted. This effect was connected with a decrease in urine and erythrocyte magnesium concentrations. Among several biochemical parameters an increase in blood glucose and lactate levels were found, leading to a decrease in blood ATP level. Supplementation of magnesium salts in inhabitants of Skawina caused a normalization of urine magnesium concentration, blood glucose and lactate levels, and increased blood ATP level, but did not diminish fluoride excretion in urine.

Comparison of pyruvate kinase variants from rat liver and Morris hepatoma 7777, obtained by an affinity chromatography on blue sepharose CL-6B.

Fractions A (salted out by ammonium sulphate between 21-30% saturation), and fractions B (salted out between 51-70% saturation) of pyruvate kinase (EC 2.7.1.40.) corresponding respectively to pyruvate kinase types L and M2 from rat liver and Morris hepatoma 7777 were purified by an affinity chromatography on Blue Sepharose CL-6B. Peaks of inactive proteins were eliminated and the enzyme fractions bound biospecifically to the gels were eluted by free ADP. The molecular mass of purified hepatoma pyruvate kinase fraction B was smaller than that of liver pyruvate kinase fraction B. Morris hepatoma pyruvate kinase fraction B represented a variant of type M2, characterised by greatest affinity to 2-phosphoenolpyruvate as a main substrate and different sensitivity to low-molecular effectors in comparison with types L from both liver and hepatoma and in comparison with type M2 from normal rat liver. Only this hepatoma fraction B showed a tumour specific sensitivity to L-cysteine and was insensitive to normal signal molecules i.e. to ATP and fructose-1,6-diphosphate which influence liver pyruvate kinase activity. L-Cysteine inhibited the tumour fraction B of pyruvate kinase by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate.

Effects of buthobendin and epinephrine on glycolytic energy formation and magnesium concentration in vitro and in vivo.

Buthobendin activated glycolytic lactate formation stronger than epinephrine and increased the ATP level, both in vitro in rabbit myocardial slices and in vivo in rabbit blood. These effects were accompanied by an increase in extracellular magnesium concentration, which was probably related to the inhibitory effect of buthobendin on the membrane-bound ATPases.

[Inhibitory effect of environmental pollutants on erythrocyte membrane ATPase activity in humans]. / Hamujacy wplyw zanieczyszczen srodowiskowych na aktywnosc ATPaz z blon erytrocytów ludzi.

In the inhabitants of Chorzów, the most polluted town in the Upper Silesia, who showed increased urine fluoride excretion and elevated blood lead concentrations, the studies on erythrocyte membrane ATPase activities have been undertaken. The investigations showed the decrease in (Na,K)ATPase activity by about 70% in comparison with the average norm in 85% of workers of the Nitrogen Chemical Plant and the Slaughterhouse (groups A and M) and by about 50% in 65% of workers of the Steelworks (group S). The decrease in Mg-ATPase activity was smaller, by about 40% in 70% persons in groups A and M, and only by 20% in comparison with the control activity in 40% persons in group S. The correlation between the decrease in (Na,K)ATPase activity in vivo and the increase in urine fluoride excretion was demonstrated. Although the possibility of lead influence on this enzyme in vivo cannot be excluded, the correlation between the changes in ATPase activity and blood lead concentration was not found.

[Mutagenicity of environmental air in Cracow monitored by the Tradescantia stamen hair system]. / Mutagennosc otaczajacego powietrza w krakowie monitorowana w ukladzie wlosków precików u Tradescantia.

Three times since 1983, groups of plants of Tradescantia clone 4430 have been exposed to ambient air on various sites of the Cracow area. The highest value of mutation frequency was noted in May 1986 after the Chernobyl accident. A slightly lower level of mutation frequency but comparable in value was riched in 1983 in vicinity to a pharmaceutical factory in result of chemical mutagen emissions. In various periods of 1987 temporary differences between the levels of mutations were observed on various sites in Cracow. There was also noted a significant decrease in the mean mutation level in comparison with the levels observed in 1983 and 1986.

[Characteristics of the population of Chorzów living in polluted health-threatening environment]. / Charakterystyka mieszkanców Chorzowa zyjacych w zanieczyszczonym, zagrazajacym zdrowiu srodowisku.

In Chorzów, the most polluted town in the Upper Silesian Industrial Region, the characteristics of three groups of inhabitants, workers of the Chemical Nitrogen Plant, the Steelworks, and the Slaughterhouse was done. Among the similar groups in respect to age and a number of males and females, the attention was paid to work, life and recreation conditions, to nutritional habits and additional customs of cigarette smoking and alcohol using. The subjective self-determination of own health was compared with objective complaints or symptoms presented in anamneses and past diseases, as well as were confronted with results of biochemical estimation of urine fluoride excretion and blood lead content. The other related results, namely magnesium concentration, urine ALA and porfobilinogen contents, erythrocyte glutathione, blood glucose, lactate and ATP levels as indices of energy and carbohydrate metabolism were presented, as well as finally (Na, K)-ATPase activity, necessary for cell membrane potential formation. The mechanisms leading to physical and psychical health depletion was demonstrated, and a new possibility of prophylactic and therapeutic actions was stressed.

[Fluorides in urine of the population living in polluted environment]. / Fluor w moczu u ludzi zyjacych w zanieczyszczonym srodowisku.

The increased excretion of fluorides in urine was shown by ion-selective method, in three different groups of workers, inhabitants of Chorzów--the most polluted town in the Upper Silesia. In nearly all workers (90%) of Nitrogen Chemical Plant and Slaughterhouse (groups A and M), the urine fluoride exceeded three to four times the upper limit of the normal range, while only two times in slightly less number of workers (77%) of the Steelworks (group S). The subjective complaints and bone pains were correlated with excessive urine fluoride concentrations in about 40% of workers studied in groups A and M, and in 18% of workers in group S. These results point to an excessive exposition of people to fluorides from the polluted environment, as well as to a high risk of fluorine retention in bones and osteofluorosis.

[Magnesium in the blood and urine of the population living in polluted environment of Chorzów]. / Magnez w krwi i w moczu ludzi zyjacych w zanieczyszczonym srodowisku Chorzowa.

In Chorzów inhabitants exposed to polluted environment, a decrease in erythrocyte and urine magnesium concentration was found in approx. 40% of workers irrespectively of type of employment. The higher percent of humans with the normal magnesium concentration in erythrocytes and urine was found in Chorzów than on the area near Skawina aluminium plant. This explained a less expressed negative effects of fluorides and lead ions in Chorzów inhabitants on energy, metabolism and health symptoms.

[Biochemical indicators of carbohydrate and energy metabolism in the Chorzów population after long-term exposure to industrial pollutants]. / Biochemiczne wskazniki przemiany weglowodanowej i energetycznej u mieszkanców Chorzowa, przewlekle narazonych na zanieczyszczenia przemyslowe.

In three groups of Chorzów inhabitants, living in the most polluted industrial environment, elevated blood lactate and glucose levels, as well as diminished blood ATP level pointed to disturbances in carbohydrate and energy metabolism. In spite of the exposition of people studied to many toxic substances from polluted environment (fluorides, lead and others), a decrease in ATP level was less expressed than in earlier investigated inhabitants of Skawina, exposed to fluorides from the Aluminium Plant, which was probably connected with greater supply of magnesium salts from food and water.