Modulation of the organization of erythrocyte membrane phospholipids by cytoplasmic ATP. The susceptibility of isoionic human erythrocyte ghosts to attack by detergents and phospholipase C

1978 ◽  
Vol 509 (1) ◽  
pp. 48-57 ◽  
Author(s):  
S.D. Shukla ◽  
M.M. Billah ◽  
R. Coleman ◽  
J.B. Finean ◽  
R.H. Michell
Keyword(s):  
Phospholipase C ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Erythrocyte Ghosts ◽  
Membrane Phospholipids

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.

Coisolation of glycophorin A and polyphosphoinositides from human erythrocyte membranes

1978 ◽  
Vol 56 (5) ◽  
pp. 349-351 ◽  
Author(s):  
J. Thomas Buckley
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Lipid Composition ◽  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Firm Conclusion ◽  
Membrane Phospholipids ◽  
Human Erythrocyte Membranes ◽  
Significant Enrichment

The lipid composition of purified erythrocyte membrane glycophorin was measured. Diphosphoinositide, triphosphoinositide, and phosphatidylserine are the major phospholipids in glycophorin preparations. Nearly all of the radioactive diphosphoinositide and triphosphoinositide extracted from erythrocyte membranes by lithium diiodosalicylate are recovered in purified glycophorin. There appeared to be no significant enrichment of other acidic membrane phospholipids in the protein. The results do not permit a firm conclusion as to whether the polyphosphoinositides are associated specifically with the membrane protein or whether fortuitous binding has occurred during purification.

scholarly journals The action of phosphatidylinositol-specific phospholipases C on membranes

1976 ◽  
Vol 154 (1) ◽  
pp. 203-208 ◽  
Author(s):  
M G Low ◽  
J B Finean
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Phosphatidyl Inositol ◽  
Detectable Effect ◽  
Erythrocyte Ghosts ◽  
Membrane Phospholipids ◽  
Liver Microsomal Fraction ◽  
Hydrolysis Of ◽  
Low Activity

A phospholipase C prepared from lymphocytes readily hydrolysed pure phosphatidyl-inositol but was relatively ineffective against phosphatidylinositol in erythrocyte “ghosts” and rat liver microsomal fraction and also against sonicated lipid extracts from these membranes. In contrast, a phospholipase C prepared from Staphylcoccus aureus readily hydrolysed phosphatidylinositol in sonicated lipid extracts but had only low activity against purified phosphatidylinositol. Unlike the enzyme from lymphocytes, the S. aureus phospholipase C did not require Ca2+ for its activity and was inhibited by cations. The previously reported specificity of this enzyme was confirmed by our observation of hydrolysis of approx. 75% of the phosphatidylinositol in ox, sheep and cat erythrocyte “ghosts” together with no detectable effect on the major erythrocyte membrane phospholipids. The phosphatidylinositol of rat liver microsomal fraction was hydrolysed only to a maximum of 15%. Some preliminary experiments showed that approx. 60% of the phosphatidylinositol of ox or sheep erythrocytes could be hydrolysed without causing substantial haemolysis.

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 INTRAMEMBRANE PARTICLE AGGREGATION IN ERYTHROCYTE GHOSTS

1974 ◽  
Vol 63 (3) ◽  
pp. 1018-1030 ◽  
Author(s):  
Arnljot Elgsaeter ◽  
Daniel Branton
Keyword(s):  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Particle Aggregation ◽  
Erythrocyte Ghosts ◽  
Intramembrane Particle ◽  
Freeze Etching ◽  
Human Erythrocyte Ghost

We have used freeze-etching and SDS-polyacrylamide gel electrophoresis to study the conditions under which the intramembrane particles of the human erythrocyte ghost may be aggregated. The fibrous membrane protein, spectrin, can be almost entirely removed from erythrocyte ghosts with little or no change in the distribution of the particles. However, after spectrin depletion, particle aggregation in the plane of the membrane may be induced by conditions which cause little aggregation in freshly prepared ghosts. This suggests that the spectrin molecules form a molecular meshwork which limits the translational mobility of the erythrocyte membrane particles.

The Phosphorus Components of Solubilized Erythrocyte Membrane Protein

1971 ◽  
Vol 49 (3) ◽  
pp. 337-347 ◽  
Author(s):  
F. B. St. C. Palmer ◽  
J. A. Verpoorte
Keyword(s):  
Sialic Acid ◽  
Membrane Protein ◽  
Organic Solvents ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Gel Electrophoresis ◽  
Electrophoretic Pattern ◽  
Erythrocyte Ghosts ◽  
Butanol Extraction ◽  
Chloroform Methanol

Membrane protein preparations were obtained by n-butanol extraction of salt-free aqueous suspensions of human erythrocyte ghosts. The solubilized protein contained 4.5% carbohydrate, including glucosamine and galactosamine, 1.7% sialic acid, and 0.2% phosphorus. Gel electrophoresis indicated the presence of a large number of proteins in the solubilized fraction. Of the phosphorus present 15% could be extracted with chloroform–methanol (2/1) and was shown to consist of phosphatidylserine and some phosphatidylinositol. A further 65% of the phosphorus was extracted with chloroform–methanol–HCl (200/100/1) and this extract was shown to consist principally of diphosphoinositide and triphosphoinositide. The remaining protein-bound phosphorus, representing 0.03% of the protein, could not be separated from the protein. Following treatment with the organic solvents the protein was resolubilized. The carbohydrate and sialic acid concentrations and the gel electrophoretic pattern were not altered. Following incubation of erythrocytes with inorganic 32P, the polyphosphoinositides were rapidly labelled. The phosphoprotein was also rapidly labelled but to a lesser extent. The phosphatidylinositol and phosphatidylserine were very poorly labelled.

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