The perturbation of the human erythrocyte membrane by phospholipase C

1975 ◽  
Vol 19 (2) ◽  
pp. 341-355
Author(s):  
A.R. Limbrick ◽  
S. Knutton
Keyword(s):  
Phospholipase C ◽  
Surface Area ◽  
Human Erythrocyte ◽  
Lipid Droplets ◽  
External Surface ◽  
Particle Density ◽  
Freeze Fracture ◽  
Erythrocyte Ghosts ◽  
Human Erythrocyte Membrane ◽  
Small Areas

A study has been made of freeze-fractured preparations of erythrocyte ghosts modified by phospholipase C (Clostridium welchii). Such membranes show a decrease in surface area of up to about 47% and lipid droplets appear on their external surface but there is no loss of protein. Freeze-fracture of maximally hydrolysed membranes exposes only very small areas of A faces and these appear particle-free. Most of the membranes are simply cross-fractured. At lower levels of hydrolysis there is more extensive exposure of A fracture faces but the particle density is less than in control preparations. If such exposed faces were representative of the whole membrane then the particle density would have been expected to increase. It is suggested either that areas of membrane with increased particle density do not fracture or that the particles revealed by freeze-fracture involve phospholipid as well as protein and are not revealed in the absence of phospholipid.

scholarly journals Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell- associated protein

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 284-289 ◽  
Author(s):  
MH Holguin ◽  
LA Wilcox ◽  
NJ Bernshaw ◽  
WF Rosse ◽  
CJ Parker
Keyword(s):  
Membrane Proteins ◽  
Phospholipase C ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Membrane Attack Complex ◽  
Human Erythrocyte Membrane ◽  
Glycosyl Phosphatidylinositol ◽  
Normal Human ◽  
Erythrocyte Membrane Proteins

Abstract The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol- specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.

scholarly journals Analysis of Integral Membrane Protein Contributions to the Deformability and Stability of the Human Erythrocyte Membrane

2001 ◽  
Vol 276 (50) ◽  
pp. 46968-46974 ◽  
Author(s):  
Heidi M. Van Dort ◽  
David W. Knowles ◽  
Joel A. Chasis ◽  
Gloria Lee ◽  
Narla Mohandas ◽  
...  
Keyword(s):  
Surface Area ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Membrane Stability ◽  
Human Erythrocyte Membrane ◽  
Lipid Interactions ◽  
Membrane Spanning ◽  
The Impact

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e.decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.

Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell- associated protein

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 284-289
Author(s):  
MH Holguin ◽  
LA Wilcox ◽  
NJ Bernshaw ◽  
WF Rosse ◽  
CJ Parker
Keyword(s):  
Membrane Proteins ◽  
Phospholipase C ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Membrane Attack Complex ◽  
Human Erythrocyte Membrane ◽  
Glycosyl Phosphatidylinositol ◽  
Normal Human ◽  
Erythrocyte Membrane Proteins

The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol- specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.

scholarly journals The organization of the major protein of the human erythrocyte membrane

1974 ◽  
Vol 137 (3) ◽  
pp. 531-534 ◽  
Author(s):  
D. H. Boxer ◽  
R. E. Jenkins ◽  
M. J. A. Tanner
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Intact Cell ◽  
The Other ◽  
Major Protein ◽  
Erythrocyte Ghosts ◽  
Human Erythrocyte Membrane ◽  
Cytoplasmic Surface ◽  
Peptide Maps

The enzyme lactoperoxidase was used to catalyse the radioiodination of membrane proteins in intact human erythrocytes and in erythrocyte ‘ghosts’. Two major proteins of the erythrocyte membrane were isolated after iodination of these two preparations, and the peptide ‘maps’ of each protein so labelled were compared. Peptides from both proteins are labelled in the intact cell. In addition, further mobile peptides derived from one of the proteins are labelled only in the ‘ghost’ preparation. Various sealed ‘ghost’ preparations were also iodinated, lactoperoxidase being present only at either the cytoplasmic or extra-cellular surface of the membrane. The peptide ‘maps’ of protein E (the major membrane protein) labelled in each case were compared. Two discrete sets of labelled peptides were consistently found. One group is obtained when lactoperoxidase is present at the extra-cellular surface and the other group is found when the enzyme is accessible only to the cytoplasmic surface of the membrane. The results support the assumption that the organization of protein E in the membrane of the intact erythrocyte is unaltered on making erythrocyte ‘ghosts’. They also confirm previous suggestions that both the sialoglycoprotein and protein E extend through the human erythrocyte membrane.

Präparative Isolierung des Protein III der menschlichen Erythrocytenmembran / Preparative Isolation of Protein III of the Human Erythrocyte Membrane

1977 ◽  
Vol 32 (1-2) ◽  
pp. 67-71 ◽  
Author(s):  
H. Schiechl
Keyword(s):  
Membrane Proteins ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Large Scale ◽  
Gel Filtration ◽  
Erythrocyte Ghosts ◽  
Human Erythrocyte Membrane ◽  
Major Intrinsic Protein ◽  
Isolation Method ◽  
Structural Modifications

Abstract The paper describes a method for the large-scale isolation of protein III (protein E, major intrinsic protein) from the human erythrocyte membrane with little expenditure of time. By treat­ment of the erythrocyte ghosts with deluted HCl at pH 2.3 and 0 °C some membrane proteins can be extracted. From the remaining “rest”- membranes, whose major constituents, besides phospholipides, are protein PAS-1, other carbohydrate containing proteins and protein III, the latter can be separated in pure form by means of gel filtration. The paper reports on the amount, purity and possible structural modifications of the protein III obtained by this isolation method.

Where’s the fat? Freeze-fracture analysis of ingredient interactions in food systems

1993 ◽  
Vol 51 ◽  
pp. 48-49
Author(s):  
Jean Fincher
Keyword(s):  
Lipid Droplets ◽  
Food Systems ◽  
Food Industry ◽  
Freeze Fracture ◽  
Food Science ◽  
Complex Interactions ◽  
Fat Reduction ◽  
Important Trend ◽  
Conventional Foods ◽  
Whipped Cream

An important trend in the food industry today is reduction in the amount of fat in manufactured foods. Often fat reduction is accomplished by replacing part of the natural fat with carbohydrates which serve to bind water and increase viscosity. It is in understanding the roles of these two major components of food, fats and carbohydrates, that freeze-fracture is so important. It is well known that conventional fixation procedures are inadequate for many food products, in particular, foods with carbohydrates as a predominant structural feature. For some food science applications the advantages of freeze-fracture preparation procedures include not only the avoidance of chemical fixatives, but also the opportunity to control the temperature of the sample just prior to rapid freezing.In conventional foods freeze-fracture has been used most successfully in analysis of milk and milk products. Milk gels depend on interactions between lipid droplets and proteins. Whipped emulsions, either whipped cream or ice cream, involve complex interactions between lipid, protein, air cell surfaces, and added emulsifiers.

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