Asoke G. Datta

Protective effect of erythropoietin on the oxidative damage of erythrocyte membrane by hydroxyl radical

Treatment of red blood cells with the copper (II) ascorbate system causes increased lipid peroxidation, increased membrane microviscosity, and phospholipid translocation with a concurrent decrease in cytosolic catalase and glutathione peroxidase activities. All these changes are prevented if the cells are treated with erythropoietin prior to the exposure to copper (II) ascorbate. The present investigation further indicates that the oxidative damage brought about by copper (II) ascorbate is due to generation of hydroxyl radical and that erythropoietin plays a unique role in protecting the membrane from oxidative damage.

Effect of erythropoietin on membrane lipid peroxidation, superoxide dismutase, catalase, and glutathione peroxidase of rat RBC

Starved animals having low levels of erythropoietin in blood showed increased MDA, fluorescent pigments, and met-Hb values whereas the hemoglobin concentration decreased significantly on starvation. In vivo and in vitro studies with Ep reversed the effects of starvation and brought these values close to normal. The activities of the enzymes (SOD, catalase, GSH-PX, GR G6PD, and 6PGD) which protect the RBC membrane directly or indirectly from peroxidative threat, decreased on starvation and restored to normal levels after Ep treatment.

Proteoliposome as the model for the study of membrane-bound enzymes and transport proteins

SummaryReconstitution of membrane-bound enzymes and transport proteins with the artificial phospholipid bilayer (liposomes) is one of the most useful techniques to study the functional aspects of these proteins. Several biochemical and biophysical parameters related to the expression of the functions of these proteins can be conveniently studied in the reconstituted proteoliposomes. Methods have been described for the preparation of model membranes and emphasis has been given specially to liposomes. Methodologies for the reconstitution of biologically active proteoliposomes using varieties of membrane-bound enzymes and proteins and their assay have been presented in details. The merits and demerits of each method and the subsequent modification of the technique have been indicated. Factors controlling the orientation and functions of the enzymes and carrier proteins in the reconstituted proteoliposomes have also been described. Attempts have been made to include few examples to describe how the reconstituted model membrane is helping us to know the molecular basis of many salient features of the biomembranes. Finally, the possible application of the proteoliposomes for the study of other complex membrane phenomena has been postulated.

Is brain a gluconeogenic organ

Glucagon increased the activities of alanine amino transferase (AAT), fructose-1∶6-bisphosphatase (fru-P2ase) and glucose-6-phosphatase (G-6-Pase) in goat brain tissue by about 100%, 150% and 50% respectively. These increase in activities were reversed by β-antagonists propranolol. Well known α-agonist and antagonist like phenylephrine and phenoxybenzamine also increased AAT and G-6-Pase activities and these increased activities were reversed by propranolol. Phenylephrine and phenoxybenzamine however did not increase brain Fru-P2ase activity.However the most interesting finding is that cerebral cortical slices could produce glucose from alanine and this glucose production was enhanced by glucagon, phenylephrine and phenoxybenzamine. Propranolol reversed the effects of these agonists and antagonist to a great extent. From all these experiments we suggest brain to be a gluconeogenic organ although much less efficient than liver.

Effect of glucagon administration on mice liver fructose-1, 6-bisphosphatase.

Intravenous administration of glucagon in mouse (200 μg/100 gm body wt), stimulated liver fructose-1,6-bisphosphatase at physiological pH by approximately 100% within 15 minutes. The stimulation was not due to protein synthesis. Similar stimulation was also observed on administration of cyclic AMP. Removal of the adrenal gland abolished the stimulatory effect of glucagon but not of cyclic AMP.

Effect of Cu2+-ascorbic acid on lipid peroxidation, Mg2+-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin

The effect of erythropoietin (Ep), a glycoprotein hormone, has been studied on lipid peroxidation induced by Cu2+ and ascorbate in vitro, Mg2+ ATPase activity and spectrin of RBC membrane. Our present investigation reveals that Cu2+ and ascorbic acid increases lipid peroxidation of RBC membrane significantly. It has further been observed that under the same experimental condition spectrin, a major cytoskeleton membrane protein, and Mg2+-ATPase activity of RBC membrane decrease significantly. However, exogenous administration of Ep completely restores lipid peroxidation and Mg2+-ATPase activity and partially recovers spectrin of RBC membrane.

Anaerobic formation of succinate from glucose and bicarbonate in resting cells of Leishmania donovani.

Abstract The cell suspension of Leishmania donovani incorporates 14 CO 2 resulting in the formation of [ 14 C]-succinic acid under anaerobic conditions. The results showed that the [ 14 C]-succinate formation from [1- 14 C]-glucose is much greater than that from [6- 14 C]-glucose. [ 14 C-pyruvate] takes part in the production of succinic acid under anaerobic conditions without decarboxylation. The anaerobic formation of succinate appears to involve the production of malate, which is then converted to succinate via the reduction of fumarate by the reversal of the tricarboxylic acid cycle. Evidence indicated that the active species in this carboxylation reaction was CO 2 although HCO 3 was active to some extent.

Effect of cadmium, mercury and copper on partially purified hepatic flavokinase of rat.

The effect of cadmium (Cd2+), mercury (Hg2+) and copper (Cu2+) was studied with partially purified flavokinase (ATP:riboflavin 5′-phosphotransferase EC 2.7.1.26) from rat liver. All the divalent heavy metal cations inhibited flavokinase activity in a concentration-dependent manner. The inhibitory effect of cadmium on the enzyme was completely reversed by increasing concentration, of Zinc (Zn2+) indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. A competition between riboflavin and Cd2+ is also evident from the present investigation. These observations hint at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. However, inhibition of flavokinase by Hg2+ could not be reversed by Zn2+. Our studies further reveal that hepatic flavokinase appears to contain an essential, accessible and functional thiol group(s) which is evident from a concentration dependent inhibition of activity by sulfhydryl reagent s like parachloromercuribenzoate (PCMB), 5,5′-dithiobis (2-nitrobenzoic acid)(DTNB), and N-ethylmaleimide (NEM). Inhibition of flavokinase by sulfhydryl reagents were protected, except in case of NEM inhibition, when the enzyme was incubated with thiol protectors like glutathione (GSH) and dithiothreitol (DTT). Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by GSH and DTT suggesting that Cd2+ probably interacts with a reactive thiol group at or near the active site of enzyme in bringing about its inhibitory effect. (Mol Cell Biochem 167: 73-80, 1997)

Effect of erythropoietin on the glucose transport of rat erythrocytes and bone marrow cells

The effect of Ep on radioactive glucose and methyl-alpha-D-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [14C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-alpha-D-glucoside inhibited the Ep-stimulated glucose transport significantly. Ep also stimulated the transport of radioactive methyl-alpha-D-glucoside which was competitively inhibited in presence of D-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-alpha-D-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa.

Effect of erythropoietin on the lipid composition of red blood cell membrane

Effect of Ep on [14C]acetate incorporation into different lipid fractions of RBC membranes in starved and phenylhydrazine-treated rats was studied. The incorporation was increased into both neutral and phospholipid fractions on Ep treatment to starved or phenylhydrazine-treated rats. A slight decrease in the ratio of neutral lipid to phospholipid was observed under the influence of Ep in starved rats (23%) or in phenylhydrazine-treated rats (36%). Incorporation of radioactivities into different phospholipid fractions of RBC membrane increased on Ep treatment to starved rats, whereas, the relative percentages of these phospholipids (except LPC) remained more or less unchanged under similar conditions. Phenylhydrazine treatment increased the relative percentage of PC and concomitantly decreased the percentage of Sph. Percentage composition of both these two phospholipids showed a tendency to return to their normal levels on administration of Ep to phenylhydrazine-treated rats. Ep decreased the sigma saturated/sigma unsaturated ratio of fatty acids in PE, PS, and PC of RBC membrane in starved rats. On the other hand, no significant change was observed in this ratio of fatty acids in the phospholipids except Sph of RBC membrane in the presence of phenylhydrazine and Ep. In Sph, the ratio went down under similar conditions.