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SOME CONSEQUENCES OF THE BINDING OF AFLATOXIN B1 WITH PLASMA MEMBRANE ON THE REGULATION OF INTRACELLULAR Ca2+ HOMEOSTASIS
Published 1992-03Call Number: Loading…
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KINETICS AND THERMODYNAMIC STUDIES OF NADP BINDING REACTIONS OF GENETIC VARIANTS OF HUMAN ERYTHROCYTE GLUCOSE 6-PHOSPHATE DEHYDROGENASE
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Page will reload when a filter is selected or excluded.- Inherited Glucose-6-Phosphate dehydrogenase (G6PD) deficiency in humans results in hemolytic anaemia. The enzyme G6PD provides a crucial link in a series of biochemical reactions which occur in the red blood cell that leads to the steady state accumulation of NADPH, reduced glutathione by glutathione reductase, and the removal of potentially dangerous organic peroxides which, if not scanvenged, may result in the formation of radical species of oxygen which damage the energy generation system, which in turn may result in swelling, lysis and hemolytic anaemia. The objective of this thesis was to investigate the human variants of the enzyme G6PD in order to provide a better understanding of the molecular basis of the enzyme activity and factors affecting the onset of the human disease. Human Erythrocyte Glucose-6- Phosphate dehydrogenase has been known to occur in many genetic variants and the catalytic active enzyme of each variant are tetramers and dimers in acidic and alkaline solution respectively. The question then is whether there would be differences in the reactivities of these variants and whether there are differences in the reactivities of the two active forms of the enzyme from the same variant. A comparative analysis of the kinetic and thermodynamic studies of NADP+ binding reactions of these variants under controlled and well-defined experimental conditions of pH and ionic strength was therefore undertaken. The binding reaction of NADP+ to G6PD B+ was also studied as a function of ionic strength of the buffers in order to evaluate the effect of these variations in the buffer system on the co-operative interactions of the NADP+ binding sites on the enzyme. The findings show that there are two binding sites on each of the enzyme variants and these were identified as imidazolium groups of histidine and sulfhydryl groups. The logKm versus pH curves show a broad plateau between pH 6.7 and 8.2 interrupted by a sharp minimum at pH 7.1 for all the enzyme variants. An explanation of this behaviour in terms of co-operative ionization of groups on the enzyme and enzyme-substrate complex which may be linked to the association - dissociation behaviour of the enzyme is proposed. In agreement with G6P binding data, the plot of the enthalpy of the dissociation of enzyme - NADP+ complex against pH shows the shape of a two U—shaped curves consistent with the existence of a tetrameric form of enzyme at acidic pH and dimeric form at alkaline pH. A similar plot of the activation energy of the reaction for each variant shows a consistent decrease of the activation energy with increase in pH, the activation energy at the pH 5.8 being almost halved at the alkaline pH of 9.0, This behaviour is explained to arise from the dimer enzyme being more reactive than the tetramer. There are two schools of thought about the existence and nature of cooperativity among the NADP+ binding sites on G6PD subunits. The study reported in this thesis has unequivocally solved the controversy between the two schools. It is now established that the tetrameric form of the enzyme shows no Cooperativity while the dimeric form is cooperative. The disagreement between the two schools of thought has been explained in the variable experimental conditions used by the workers in the two schools. We have shown that an experimental condition that favours tetramer formation therefore favours non-cooperativity while a condition that favours dimer formation favours cooperativity. The inhibition study by primaquine phosphate shows a complex interaction of this effector with G6PD. There is activation of the G6PD activity at low effector concentration and inhibition at high concentration. This interaction may be due to oxidation of NADPH at low primaquine concentration resulting in generation of more NADP+ which increases the activity of the enzyme. Such a situation might account for the increased hemolysis in variant subjects with low Artracellular NADPH concentration which will result in low level of reduced glutathione. Reduced glutathione is necessary for the maintenance of the integrity of the red cells. 1 results 1
- The possible influence of aflatoxin B1 a potent hepatocellular carcinogen on the regulation of intracellular Ca2+ homeostasis has been studied using the red cell as a model. Preliminary work on the interaction of the toxin with the red cell membrane using spectrofluometric analysis indicated that the toxin binds spontaneously and irreversibly to the red cell membrane. The binding is highest at pH 4 and least at pH 10. Results obtained from studies using equilibrum dialysis technique show that about 4 nmoles of the toxin bind to one microgram membrane protein. Although the exact membrane component to which aflatoxin B1 binds is not known, experiments carried out to determine the influence of aflatoxin B1 on the activity of the calcium pumping protein revealed that the toxin inhibited the calmodulin-stimulated erythrocyte membrane Ca2+ -ATPase activity by about 50 percent, while it has little or no effect on its basal activity. Kinetic analysis of the inhibition shows that, the toxin reduces the Vmax and Km of the calmodulin-stimulated enzyme by 50 percent in a non-competitive manner, On the other hand, the carcinogen had no significant influence on the kinetic parameters of the enzyme in the non-activated state. Similar results were obtained for the triton X-100 solubilized and calmodulin affinity chromatographed enzyme. In this instance aflatoxin B1 inhibited the calmodulin-stimulated purified enzyme by 50 percent with or without preincubation on ice for half an hour. Again, the toxin had little or no effect on the basal activity of the enzyme in the absence of calmodulin. Analysis of the results obtained using varying concentrations of ATP shows that the Km and Vmax of the non-activated enzyme were not altered by the toxin while both the Vmax and Km values were reduced by about 50 percent in the presence of calmodulin. In addition aflatoxin B1 inhibited Diphosphotidyl glycerol (cardiolipin) by about 28% while it has no effect on the basal activity of the enzyme. Although, the inhibition of the membrane bound or purified Ca2+ ATPase by the toxin is concentration dependent, varying concentrations of phosphatidyl serine and phosphatidyl choline do not affect the inhibition of the purified enzyme by afla toxin B1. Results obtained with triton X-100 solubilized enzyme shows that triton X-100 alone could not activate the enzyme. Thus at triton X-100: protein ratio of 2, the enzyme was stimulated by calmodulin. This activity was sensitive to inhibition by the toxin. In this instance, the calmodulin-stimulated activity was inhibited by about 50%, while at lower ratios of the triton X-100 to protein there was no significant inhibition of enzyme. Results of experiments carried out on the 124KDa fragment, which was produced as a result of exposure to calpain a Ca2+ - dependent cysteine protease, indicated that the toxin has no effect whatsoever on the activity of the fragmented enzyme, Similarly experiments on limited proteolysis of the Ca2+ ATPase by trypsin to give the 90KDa fragment which still retains its calmodulin binding domain and the 76KDa fragment which has lost its calmodulin binding domain revealed that the aflatoxin inhibited the 90KDa fragment by about 50% while the 76KDa fragment is not affected at all. Altogether, -these findings show that aflatoxin B1 inhibits the plasma membrane Ca2+ - pumping ATPase by interacting with the enzyme at the calmodulin binding domain. The nature of the exact amino acid residue to which the toxin binds is however not known. The implication of these observations is that Ca2+ extrusion may be hampered in situations where the cell is poisoned by the aflatoxin 1 results 1