The membrane abnormality of the red cell in paroxysmal nocturnal haemoglobinuria

P. G. Righetti, M. Perrella, A. Zanella, G. Sirchia

Research output: Chapter in Book/Report/Conference proceedingChapter

15 Citations (Scopus)

Abstract

In paroxysmal nocturnal hemoglobinuria (PNH) a unique membrane abnormality renders the red blood cell (RBC) extremely sensitive to the lytic action of complement. The nature of this abnormality is still unknown. While an alteration of the cell lipids had been postulated previously, recent studies located the abnormality on the membrane proteins. The sulphydryl reagent 2 amino ethyl isothiouronium bromide (AET) is one of the most effective compounds in producing a PNH like abnormality in normal RBC's. The authors studied the membrane proteins of normal, PNH and AET RBC's by sodium dodecyl sulphate (SDS) disc electrophoresis in polyacrylamide gel slabs. A 100-400 μg membrane pellet is dissolved in 2% SDS, containing 10% glycerol, 5% 2 mercaptoethanol and 0.001% bromophenol blue in 0.0625M Tris HCl buffer, pH 6.8. After 3-5 min boiling, the samples are immediately cooled in ice and loaded in 7.5% acrylamide, 0.1% SDS gel slabs. After 1 hr at 5W, the electrophoresis is continued for another 6 hr at 50 mA and 200 V. By this technique, it is demonstrated that a 81,700 ± 600 dalton chain, present in normal ghosts, is lacking in PNH ghosts, where a new 77,100 ± 600 dalton polypeptide appears. The same lesion is produced by a brief exposure of normal cells to AET: thus the 77,100 dalton chain in PNH ghosts is not genetically determined, but is a degradation product of the normal 81,700 dalton chain. When normal and abnormal ghosts are mixed before denaturation in SDS, their SDS gel pattern is additive: this rules out degradation due to proteolytic enzymes during sample manipulation. The lesion is extensive, since it affects approximately 5x105 chains/ghost (10% of the total membrane proteins). The degraded protein is the major glycoprotein of the RBC membrane. The 77,100 chain is still Schiff positive: probably what is released is a small glycopeptide fragment. These findings were recently confirmed on homogeneous glycoprotein preparations from normal, PNH and AET cells. A direct effect of AET on the glycoprotein is excluded; an indirect effect, for instance the activation of a protease buried in the cell membrane, is postulated. Even if the protease activation hypothesis is accepted, its occurrence in PNH cells remains to be explained. (Righetti - Milan).

Original languageEnglish
Title of host publicationNATURE NEW BIOL.
Pages273-275
Number of pages3
Volume245
Edition148
Publication statusPublished - 1973

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Paroxysmal Hemoglobinuria
beta-Aminoethyl Isothiourea
Sodium Dodecyl Sulfate
Membranes
Glycoproteins
Membrane Proteins
Peptide Hydrolases
Isothiuronium
Bromphenol Blue
Erythrocytes
Gels
Cell Membrane
Disc Electrophoresis
Tromethamine
Glycopeptides
Mercaptoethanol
Acrylamide
Ice
Bromides
Glycerol

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Righetti, P. G., Perrella, M., Zanella, A., & Sirchia, G. (1973). The membrane abnormality of the red cell in paroxysmal nocturnal haemoglobinuria. In NATURE NEW BIOL. (148 ed., Vol. 245, pp. 273-275)

The membrane abnormality of the red cell in paroxysmal nocturnal haemoglobinuria. / Righetti, P. G.; Perrella, M.; Zanella, A.; Sirchia, G.

NATURE NEW BIOL.. Vol. 245 148. ed. 1973. p. 273-275.

Research output: Chapter in Book/Report/Conference proceedingChapter

Righetti, PG, Perrella, M, Zanella, A & Sirchia, G 1973, The membrane abnormality of the red cell in paroxysmal nocturnal haemoglobinuria. in NATURE NEW BIOL.. 148 edn, vol. 245, pp. 273-275.
Righetti PG, Perrella M, Zanella A, Sirchia G. The membrane abnormality of the red cell in paroxysmal nocturnal haemoglobinuria. In NATURE NEW BIOL.. 148 ed. Vol. 245. 1973. p. 273-275
Righetti, P. G. ; Perrella, M. ; Zanella, A. ; Sirchia, G. / The membrane abnormality of the red cell in paroxysmal nocturnal haemoglobinuria. NATURE NEW BIOL.. Vol. 245 148. ed. 1973. pp. 273-275
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N2 - In paroxysmal nocturnal hemoglobinuria (PNH) a unique membrane abnormality renders the red blood cell (RBC) extremely sensitive to the lytic action of complement. The nature of this abnormality is still unknown. While an alteration of the cell lipids had been postulated previously, recent studies located the abnormality on the membrane proteins. The sulphydryl reagent 2 amino ethyl isothiouronium bromide (AET) is one of the most effective compounds in producing a PNH like abnormality in normal RBC's. The authors studied the membrane proteins of normal, PNH and AET RBC's by sodium dodecyl sulphate (SDS) disc electrophoresis in polyacrylamide gel slabs. A 100-400 μg membrane pellet is dissolved in 2% SDS, containing 10% glycerol, 5% 2 mercaptoethanol and 0.001% bromophenol blue in 0.0625M Tris HCl buffer, pH 6.8. After 3-5 min boiling, the samples are immediately cooled in ice and loaded in 7.5% acrylamide, 0.1% SDS gel slabs. After 1 hr at 5W, the electrophoresis is continued for another 6 hr at 50 mA and 200 V. By this technique, it is demonstrated that a 81,700 ± 600 dalton chain, present in normal ghosts, is lacking in PNH ghosts, where a new 77,100 ± 600 dalton polypeptide appears. The same lesion is produced by a brief exposure of normal cells to AET: thus the 77,100 dalton chain in PNH ghosts is not genetically determined, but is a degradation product of the normal 81,700 dalton chain. When normal and abnormal ghosts are mixed before denaturation in SDS, their SDS gel pattern is additive: this rules out degradation due to proteolytic enzymes during sample manipulation. The lesion is extensive, since it affects approximately 5x105 chains/ghost (10% of the total membrane proteins). The degraded protein is the major glycoprotein of the RBC membrane. The 77,100 chain is still Schiff positive: probably what is released is a small glycopeptide fragment. These findings were recently confirmed on homogeneous glycoprotein preparations from normal, PNH and AET cells. A direct effect of AET on the glycoprotein is excluded; an indirect effect, for instance the activation of a protease buried in the cell membrane, is postulated. Even if the protease activation hypothesis is accepted, its occurrence in PNH cells remains to be explained. (Righetti - Milan).

AB - In paroxysmal nocturnal hemoglobinuria (PNH) a unique membrane abnormality renders the red blood cell (RBC) extremely sensitive to the lytic action of complement. The nature of this abnormality is still unknown. While an alteration of the cell lipids had been postulated previously, recent studies located the abnormality on the membrane proteins. The sulphydryl reagent 2 amino ethyl isothiouronium bromide (AET) is one of the most effective compounds in producing a PNH like abnormality in normal RBC's. The authors studied the membrane proteins of normal, PNH and AET RBC's by sodium dodecyl sulphate (SDS) disc electrophoresis in polyacrylamide gel slabs. A 100-400 μg membrane pellet is dissolved in 2% SDS, containing 10% glycerol, 5% 2 mercaptoethanol and 0.001% bromophenol blue in 0.0625M Tris HCl buffer, pH 6.8. After 3-5 min boiling, the samples are immediately cooled in ice and loaded in 7.5% acrylamide, 0.1% SDS gel slabs. After 1 hr at 5W, the electrophoresis is continued for another 6 hr at 50 mA and 200 V. By this technique, it is demonstrated that a 81,700 ± 600 dalton chain, present in normal ghosts, is lacking in PNH ghosts, where a new 77,100 ± 600 dalton polypeptide appears. The same lesion is produced by a brief exposure of normal cells to AET: thus the 77,100 dalton chain in PNH ghosts is not genetically determined, but is a degradation product of the normal 81,700 dalton chain. When normal and abnormal ghosts are mixed before denaturation in SDS, their SDS gel pattern is additive: this rules out degradation due to proteolytic enzymes during sample manipulation. The lesion is extensive, since it affects approximately 5x105 chains/ghost (10% of the total membrane proteins). The degraded protein is the major glycoprotein of the RBC membrane. The 77,100 chain is still Schiff positive: probably what is released is a small glycopeptide fragment. These findings were recently confirmed on homogeneous glycoprotein preparations from normal, PNH and AET cells. A direct effect of AET on the glycoprotein is excluded; an indirect effect, for instance the activation of a protease buried in the cell membrane, is postulated. Even if the protease activation hypothesis is accepted, its occurrence in PNH cells remains to be explained. (Righetti - Milan).

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