Solubilization of human erythrocyte membranes by ASB detergents

Domingues, Cleyton C.; Malheiros, Sônia Valéria Pinheiro; Paula, Eneida de

doi:10.1590/s0100-879x2008000900003

Cited by 26 publications

(25 citation statements)

References 32 publications

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“…Erythrocyte ghosts were prepared by sequential lyses of RBC VL using 5.0 mM, 2.5 mM and 1.25 mM ice-cold phosphate buffer, pH 7.0. The ghost membranes were solubilized in solubilizing buffer containing Tris-HCl (0.05 M), 1% (w/v) detergent concentration (CHAPS∶BOG 1∶1), MgCl 2 (1.0 mM), CaCl 2 (1.0 mM), dithiothreitol (DTT, 0.2 mM), phenylmethyl sulfonyl fluoride (PMSF, 20 µg/L), protease inhibitor cocktail, pH 7.2, sonicated (three pulses, 10 sec each) in ice-mixture and incubated at 4°C for 1 hr [43]. After centrifugation at 8200×g, 4°C the supernatant was collected and dialyzed against Tris-HCl (0.05 M, pH 7.2) saline (TBS) containing 0.03 M Ca 2+ (TBS-Ca 2+ ), 0.01% (w/v) detergent (CHAPS∶ BOG 1∶1), sodium azide (0.02%).…”

Section: Methodsmentioning

confidence: 99%

Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

et al. 2011

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Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in α2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBCVL). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic α- and β-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrinVL) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to α- and β-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrinN) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrinN. Although the presence of both N- and O-glycosylations was found both in spectrinN and spectrinVL, enhanced sialylation was predominantly induced in spectrinVL. Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of α- and β-spectrinVL confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrinVL showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrinN suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBCVL. The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrinVL as evidenced by the presence of an additional 60 kDa fragment, absent in spectrinN which possibly affects the biology of RBCVL linked to both severe distortion of erythrocyte development and impairment of erythrocyte membrane integrity and may provide an explanation for their sensitivity to hemolysis and anemia in VL patients.

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Section: Methodsmentioning

confidence: 99%

Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

et al. 2011

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“…This experimental model have been used to study the influence of solutes such as ethanol, glycerol and sorbitol [7,8], natural products [24], detergents [25][26][27], and degenerative processes [28][29][30] on the stability of erythrocyte membrane.…”

Section: Introductionmentioning

confidence: 99%

Influence of Glucose Concentration on the Membrane Stability of Human Erythrocytes

Lemos

Márquez-Bernardes

Arvelos

et al. 2011

Cell Biochem Biophys

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The action of glucose as an osmolyte in relation to blood cells is not well-characterized in the literature. This study aimed to study the influence of glucose concentration on the stability of red blood cells. The stability of erythrocytes was evaluated by the half-transition point obtained from the curves of lysis induced by glucose in the absence of salt or by increase in medium hypotonicity in the absence and the presence of different concentrations of glucose. In the presence of 0.9 g/dl NaCl, there was no hemolysis with increasing concentration of glucose from 0 to 10 g/dl. In the absence of NaCl, the dependence of hemolysis with the 0-10 g/dl glucose was described by a decreasing sigmoid, with fully lysed and fully protected cells being encountered in the presence of 0-2 and 4-10 g/dl glucose, respectively. The possible origin of such stabilization effect is discussed with base of what is known about osmostabilization of biological complexes and about the influence of glucose on the rheological properties of erythrocytes.

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“…Fig. 2) d According to K.cmc parameter (Heerklotz and Seelig 2000) e According to R e sat f Roda et al 1983 g Campos et al 2012 concentration for micelle formation (Domingues et al 2008). For instance, the D w value found for TX-100 (51 μM, Preté et al 2002a) is, as expected, smaller than its cmc (Table 1).…”

Section: Isosmotic Hemolysis and Parameters To Describe Detergent Solmentioning

confidence: 61%

“…In 1985, Lichtenberg proposed a quantitative approach to analyze the lipid-bilayer disruption induced by detergents (Lichtenberg 1985). The approach was further employed to describe detergent-induced hemolysis by the group of Goñi (Partearroyo et al 1992(Partearroyo et al , 1996Requero et al 1995) and ours Preté et al 2002a, b;Domingues et al 2008).…”

Section: Isosmotic Hemolysis and Parameters To Describe Detergent Solmentioning

confidence: 99%

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity

et al. 2017

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Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose R e sat -the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C 12 E 8 and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.

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Solubilization of human erythrocyte membranes by ASB detergents

Cited by 26 publications

References 32 publications

Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

Influence of Glucose Concentration on the Membrane Stability of Human Erythrocytes

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity

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