Inhibition of complement by a substance isolated from human erythrocytes—II , Studies on the site and mechanism of action

Hoffmann, Edward M.

doi:10.1016/0161-5890(69)90071-6

Cited by 24 publications

(28 citation statements)

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“…After the last wash, the ghosts were resuspended to 2.5 ml in ghosting buffer. Next, 3 M NaCl was added to the samples so that the final concentration was 150 mM (24,25). An aliquot of the samples was reserved for determination oftotal protein, and n-butanol (20%o vol/vol) was added to the remainder of the two suspensions (24,25).…”

Section: Methodsmentioning

confidence: 99%

“…Next, 3 M NaCl was added to the samples so that the final concentration was 150 mM (24,25). An aliquot of the samples was reserved for determination oftotal protein, and n-butanol (20%o vol/vol) was added to the remainder of the two suspensions (24,25). The samples were stirred for 15 min at room temperature and then centrifuged at 40C for 10 min at 48,000 g. The butanol saturated aqueous phase was recovered and dialyzed extensively against VBS.…”

Section: Methodsmentioning

confidence: 99%

“…After 30 min, 4 ml of cold GVB-EDTA were added, and the reaction mixtures were centrifuged at 1,000 g for 3 min. Hemolysis was subsequently quantified by measuring free hemoglobin (aliquots 13.25 mS at RT by the addition of 3 M NaCl (24,25). Next, n-butanol was added (20% vol/vol) and the mixture was stirred at room temperature for 20 min (24,25).…”

Section: Methodsmentioning

confidence: 99%

“…Hemolysis was subsequently quantified by measuring free hemoglobin (aliquots 13.25 mS at RT by the addition of 3 M NaCl (24,25). Next, n-butanol was added (20% vol/vol) and the mixture was stirred at room temperature for 20 min (24,25). After centrifugation at 10,000 g for 25 min, the butanol saturated aqueous phase (1,280 ml containing 106 pg/ml of protein) was recovered and dialyzed extensively against 20 mM sodium phosphate, 20 mM NaCG, pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

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Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

Holguin¹,

Fredrick²,

Bernshaw³

et al. 1989

J. Clin. Invest.

367

202

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The observation that type MI erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are susceptible to hemolysis initiated by activated cobra venom factor complexes (CoFBb), whereas normal erythrocytes are resistant, implies that the PNH III cells are deficient in a membrane constituent that regulates this process. To isolate the inhibitory factor from normal erythrocytes, membrane proteins were first extracted with butanol and then subjected to sequential anion exchange, hydroxylapatite, and hydrophobic chromatography. Analysis by SDS-PAGE and silver stain of the inhibitory fractions showed a single band corresponding to a protein with an apparent Mr of 18 kD. PNH erythrocytes were incubated with incremental concentrations of the radiolabeled protein and then washed. In a dose-dependent fashion, the protein incorporated into the cell membrane and inhibited CoFBb-initiated lysis. This protein inhibitor functioned by restricting the assembly of the membrane attack complex at the level of C7 and CB incorporation. By using a monospecific antibody to block the function of the inhibitor, it was shown that normal erythrocytes are rendered susceptible to CoFBb-initiated hemolysis. Analysis by Western blot of membrane proteins revealed that PNH Iml erythrocytes are deficient in the 18-kD protein. By virtue of its molecular weight and inhibitory activity, the 18-ID protein appears to be discrete from other previously described erythrocyte membrane proteins that regulate complement. These studies also indicate that the susceptibility of PNH III erythrocytes to reactive lysis is causally related to a deficiency of the 18-kD membrane inhibitor.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

Holguin¹,

Fredrick²,

Bernshaw³

et al. 1989

J. Clin. Invest.

367

202

View full text Add to dashboard Cite

show abstract

“…28,29 He also showed that a portion of the active material possessed the capacity to bind to the indictor cell and retain functional activity. Hoffmann used the name decay accelerating factor (DAF) to describe the functional property of the extract because the material enhanced the rate at which the complement C3 convertase diminished over time.…”

Section: Identification Of the Erythrocyte Membrane Proteins That Regmentioning

confidence: 99%

Paroxysmal Nocturnal Hemoglobinuria: An Historical Overview

Parker¹

2008

Hematology

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The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is episodic hemoglobinuria, and it was this feature that captured the attention of European physicians in the latter half of the 19th century, resulting in careful observational studies that established PNH as an entity distinct from paroxysmal cold hemoglobinuria and march hemoglobinuria. Curiosity about the etiology of the nocturnal aspects of the hemoglobinuria led the German physician Paul Strübing to develop the prescient hypothesis that the erythrocytes of PNH are abnormally sensitive to hemolysis when the plasma is acidified during sleep because of accumulation of carbon dioxide and lactic acid as a result of slowing of the circulation. Investigation of the intricate pathophysiology that underlies the abnormal sensitivity of PNH erythrocytes to hemolysis in acidified serum produced a number of remarkable scientific achievements that involved discovery of the alternative pathway of complement, identification of the membrane proteins that regulate complement, discovery of a novel mechanism for attachment of proteins to the cell surface, and identification of the genetic basis of the disease. These discoveries were made steadily over a period of more than 100 years, and each generation of physicians and scientists made important contributions to the field. The mysteries of PNH have been solved in a particularly satisfying way because the precision and orderliness of the solutions made clearly understandable what had seemed at the times prior to resolution to be problems of nearly insurmountable complexity. The history of PNH is an inspirational reminder of the elegant complexity of nature, the rewards of curiosity and the power and beauty of science.

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Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)

Parker

1994

CP in Immunology

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The complement system is the primary effector of humoral immunity. Because of its enormous destructive capacity, mechanisms for confining the activity of the system to the desired target and elaborate safeguards for protecting self against complement-mediated injury have evolved. Human cells, particularly those found at sites of inflammation (e.g., hematopoietic and endothelial cells), express highly specialized membrane constituents that act independently or in concert with plasma regulatory proteins to inhibit the functional activity of complement. Decay-accelerating factor (DAF), or CD55, directly inhibits the formation and stability of the amplification C3 and C5 convertases of both the classical and the alternative pathways. Failure of a cell to regulate the amplification C3 and C5 convertases allows the generation of the potentially cytolytic membrane attack complex (MAC), or C5b-9 (consisting of the complement components C5b, C6, C7, C8, and C9). The primary cellular regulator of the MAC is the membrane inhibitor of reactive lysis (MIRL), or CD59, which restricts complement-mediated lysis by blocking assembly of the MAC (primarily at the stage of C9 binding and polymerization). This unit provides a basic protocol for isolating CD55 and CD59, along with two support protocols describing separate functional assays for CD59 and CD55.

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Inhibition of complement by a substance isolated from human erythrocytes—II , Studies on the site and mechanism of action

Cited by 24 publications

References 0 publications

Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

Paroxysmal Nocturnal Hemoglobinuria: An Historical Overview

Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)

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