Intact Rabbit Erythrocytes Research Articles

Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.

Isolation and characterization of a human alternative complement pathway-inhibiting protein from larval hemolymph of the silkworm, Bombyx mori

An alternative complement pathway-inhibiting protein (ACPIP), which inhibits the activation of the alternative complement pathway (ACP) of the human serum, was isolated from larval hemolymph of the silkworm, Bombyx mori, by using ammonium sulfate fractionation and column chromatographies to homogeneity. About 400 μg of ACPIP was routinely obtained from 20 ml hemolymph. The purified ACPIP preparation consisted of two distinct polypeptides (34 and 32 kDa) on SDS-PAGE. The amino acid compositions of the two polypeptides were nearly identical; 21% of the amino acid residues were acidic. The amino terminal amino acid sequences up to 20 residues in these two polypeptides were also identical. Addition of the ACPIP to human serum resulted in a dose-dependent inhibition of the hemolysis of intact rabbit erythrocytes via the ACP, whereas in no inhibition of hemolysis of sensitized-sheep erythrocytes (EA) via the classical pathway.

Agents facilitating the electric field-induced fusion of intact rabbit erythrocytes

In the recent years, the role of specific membrane active agents in the electrofusion process has started to draw attention, and it has been found that the presence of various substances in the cell medium can affect the fusion process either in a positive or negative way. In this work, the effect of several proteins, bivalent cations and antibiotics was tested with respect to their ability to protect intact erythrocytes from hemolysis and facilitate the fusion process. The effect of different sugars was also studied. Among the different proteins, pronase and proteinase were found to be the most effective. With respect to bivalent cations, Ca 2+ and Mn 2+ were more effective while Mg 2+ was less important. From the antibiotics, penicillin caused a negative effect while streptomycin acted positively. Finally, glucose medium was found to be the most effective compared to all sugars tested. The results indicated that there are strong differentiations of the induced effects caused by each substance, and some possible mechanisms of action of these agents on the erythrocyte membrane were discussed.

Substrates of Hexokinase, Glucose-6-Phosphate Dehydrogenase, and Glyceraldehyde-3-Phosphate Dehydrogenase Prevent the Inhibitory Response Induced by Ascorbic Acid/Iron and Dehydroascorbic Acid in Rabbit Erythrocytes

Exposure of intact rabbit erythrocytes or erythrocyte lysates to ascorbic acid/FeCl3in a glucose-free saline promoted a rapid decline in reduced glutathione and this response was paralleled by inactivation of hexokinase. Under the same conditions, the activity of the enzymes glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase did not show appreciablevariations in intact cells, but was severely inhibited in the cell-free system. Similar results were obtained by replacing ascorbic acid/FeCl3with dehydroascorbic acid. In addition, both treatments effectively inhibited the activity of purified hexokinase as well as those of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Further studies using the cell-free system indicated that the inhibition of enzyme activities elicited by either of the two treatments was effectively counteracted by the specific substrates of these enzymes. The fact that the hexokinase substrate glucose freely permeates the plasma membrane, unlike the substrates of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphatedehydrogenase, explains the selective inhibition of hexokinase observed in intact cells. The above results also indicate that dehydroascorbic acid is an inhibitor of these enzymes and strongly suggest that it is at least in part responsible for the effects mediated by the cocktail ascorbic acid/FeCl3.

Regulation of NAD+ glycohydrolase activity by NAD(+)-dependent auto-ADP-ribosylation.

NAD+ glycohydrolase (NADase; EC 3.2.2.5) is an enzyme that catalyses hydrolysis of NAD+ to produce ADP-ribose and nicotinamide. Its physiological role and the regulation of its enzymic activity have not been fully elucidated. In the present study, the mechanism of self-inactivation of NADase by its substrate, NAD+, was investigated by using intact rabbit erythrocytes and purified NADase. Our results suggest that inactivation of NADase was due an auto-ADP-ribosylation reaction. ADP-ribosylated NADase of rabbit erythrocytes was deADP-ribosylated when incubated without NAD+, and thus enzyme activity was simultaneously restored. These findings suggest that reversible auto-ADP-ribosylation of NADase might regulate the enzyme's activity in vivo.

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23±1.90 μmol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg·ATP 2−. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 μM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP 4− and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94±0.02 μmol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92±0.3 μmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.

Phorbol ester stimulates the phosphorylation of rabbit erythrocyte band 4.1

The effect of phorbol esters on membrane phosphorylation was examined in intact rabbit erythrocytes prelabeled with [ 32P ]orthophosphate. Tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate, but not the inactive 4α-phorbol 12,13-didecanoate, specifically stimulated the phosphorylation of two proteins in the erythrocyte membrane. They have apparent molecular weight of 100,000 dalton and 85,000 dalton. When intracellular calcium concentrations were raised by ionomycin, A23187, both the 85,000 dalton polypeptide and the 85K phosphoprotein were degraded. The 85,000 dalton phosphoprotein showed cross-reactivity with antiserum to human erythrocyte band 4.1. Based on its susceptibility to calcium-activated protease and its immunological property, the 85,000 dalton phosphoprotein was identified to be the band 4.1 of the erythrocyte membrane skeletal network.

The role of hemoglobin in the N-oxidation of 4-chloroaniline

Rabbit hemoglobin effects reduced pyridine nucleotide-dependent N-oxidation of 4-chloroaniline in the presence of NADPH-cytochrome c ( P-450) reductase (EC 1.6.2.4). The reaction is blocked by the addition of CO, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6). The apparent K m value for the amine is 5.9 mM. the substrate interacts with hemoglobin in a non-cooperative manner; highly purified α- and β-subunits mediate amine oxidation with kinetic constants close to those of the intact tetramer. Metabolism of 4-chloroaniline is associated with the formation of a 421 nm absorbing spectral complex, which might represent a ferryl species or a product adduct. Rapid reaction measurements suggsts that either transfer of the second electron or product dissociation limits the overall rate of hemoglobin cycling. Erythrocyte reductases, such as ‘NADPH-methemoglobin reductase’ or soluble NADH-cytochrome b 5 reductase (EC 1.6.2.2), also sustain amine oxidation in the presence of an appropriate electron carrier. Similarly, intact rabbit erythrocytes generate low amounts of N-oxy product when incubated with the parent amine. These findings support the notion that the red blood cell might be a site of bioactivation of aromatic amines, some of which, after being N-oxidized, become potent mutagens and carcinogens.

Phosphorus-31 spectroscopic determinations of the phosphorus metabolite profiles of blood components: Erythrocytes, reticulocytes, and platelets

The phosphorus-31 nuclear magnetic resonance spectroscopic profiles of intact rabbit erythrocytes, reticulocytes, and human platelets have been obtained and compared, and the observed resonances identified with known phosphorus-containing compounds. The relative levels of low- and high-energy phosphates in the spectra have been correlated with the metabolic states of these blood components. Spectra of perchloric acid extracts of these tissues have been compared to those from the intact states. Two hitherto unknown resonances have been seen in the orthophosphate diester region of the spectra of platelets and reticulocytes, but not in mature erythrocytes. It was also observed that ferricyanide ion was a suitable substrate for maintaining the viability of reticulocytes in the anaerobic NMR system. Platelets stored at 4° had considerably lower metabolic reserves than either fresh platelets or those stored at room temperature, which showed only slightly diminished stores of high-energy phosphates, in accord with their known survival characteristics. It was concluded from the study that 31P spectroscopy will be a valid analytical probe in the study of intact blood components in any disease which alters their phosphorus-containing metabolites.

The role of sialic acid in the determination of survival of rabbit erythrocytes in the circulation

Evidence is presented to indicate a generalized role for the terminal sialic acid residues of circulating erythrocytes of rabbit. Neuraminidase is shown to remove only sialic acid from these erythrocytes. Neuraminidase-treated and intact rabbit erythrocytes have similar in vitro properties, except those of cellular charge and cellular adhesion in their sera. These properties include similar shape, osmotic fragility curve, autohemolysis at 37°, K + retention and pyruvate kinase activity. The D-glucose 6-phosphate dehydrogenase and the cholinesterase activities are higher on the neuraminidase-treated erythrocytes than on the intact ones. After injection into rabbits, the sialic acid-less erythrocytes tested, were promptly removed from the circulation; intact erythrocytes, previously incubated under the same conditions but without neuraminidase, were removed from the circulation after a significantly longer period.