scholarly journals Isolation and characterization of the complement-inhibiting protein CD59 antigen from platelet membranes

1992 ◽  
Vol 282 (2) ◽  
pp. 409-413 ◽  
Author(s):  
B P Morgan
Keyword(s):  
Functional Characterization ◽  
Membrane Attack Complex ◽  
Cell Types ◽  
Erythrocyte Membranes ◽  
Glycosyl Phosphatidylinositol ◽  
Platelet Protein ◽  
Isolation And Characterization ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes ◽  
Inhibiting Protein

Several groups have recently described the isolation of a 20 kDa membrane-attack-complex (MAC)-inhibiting protein, termed ‘CD59 antigen’, from human erythrocyte membranes. Antibodies raised against erythrocyte CD59 antigen detect antigen on the surface of many other cell types, and in some of these cells the antigen has been shown to have a molecular mass similar to that of the erythrocyte protein and to confer resistance to lysis by the MAC. A platelet-membrane form of CD59 antigen has been described and reported to be much larger than the erythrocyte protein. Here I report the isolation of CD59 antigen from platelet membranes and its molecular and functional characterization. The platelet protein is not significantly larger than the erythrocyte form and possesses similar MAC-inhibiting activity. Platelet CD59 antigen is anchored to the membrane via a glycosyl-phosphatidylinositol link, and consequently it is suggested that deficiency of this protein might be responsible for the increased thrombotic tendency observed in paroxysmal nocturnal haemoglobinuria.

scholarly journals Isolation and characterization of a membrane-attack-complex-inhibiting protein present in human serum and other biological fluids

1990 ◽  
Vol 265 (2) ◽  
pp. 471-477 ◽  
Author(s):  
M J Watts ◽  
J R Dankert ◽  
B P Morgan
Keyword(s):  
Cerebrospinal Fluid ◽  
Polyclonal Antibodies ◽  
Biological Fluids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Isolation And Characterization ◽  
Human Erythrocyte Membranes ◽  
Inhibiting Protein

We have previously reported the isolation of a membrane-attack-complex-inhibiting protein (MIP) from human erythrocyte membranes [Watts, Patel & Morgan (1987) Complement 4, 236] and the production of polyclonal antibodies to this protein. Here we report the identification in plasma, urine, saliva and cerebrospinal fluid of a protein immunochemically identical with the membrane-derived MIP. The protein has been isolated from plasma by immunoaffinity chromatography on an anti-(erythrocyte MIP)-Sepharose column and shown by SDS/polyacrylamide-gel electrophoresis to be of similar molecular mass to the erythrocyte protein (55 kDa non-reduced and 65 kDa under reducing conditions). Monoclonal antibodies have been raised against plasma MIP and used to establish a two-site enzyme-linked immunoadsorbent assay, enabling quantification of MIP in plasma, urine and cerebrospinal fluid. Plasma MIP, though not able to incorporate spontaneously into membranes, was deposited on heterologous and homologous erythrocyte membranes during complement activation in a C8-dependent manner. Depletion of MIP from plasma resulted in enhancement of the lytic capacity of the plasma on heterologous erythrocytes.

scholarly journals Interaction of platelet plasma membranes with thrombin-activated platelets

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 305-312 ◽  
Author(s):  
HR Prasanna ◽  
HH Edwards ◽  
DR Phillips
Keyword(s):  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Erythrocyte Membranes ◽  
Functional Sites ◽  
Activated Platelets ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes

Abstract This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

scholarly journals Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement

1992 ◽  
Vol 284 (1) ◽  
pp. 169-176 ◽  
Author(s):  
T R Hughes ◽  
S J Piddlesden ◽  
J D Williams ◽  
R A Harrison ◽  
B P Morgan
Keyword(s):  
Affinity Purification ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Inhibitory Protein ◽  
Rat Erythrocytes ◽  
Butanol Extract ◽  
Isolation And Characterization ◽  
Membrane Bound

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated ‘reactive’ lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

scholarly journals Interaction of platelet plasma membranes with thrombin-activated platelets

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 305-312
Author(s):  
HR Prasanna ◽  
HH Edwards ◽  
DR Phillips
Keyword(s):  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Erythrocyte Membranes ◽  
Functional Sites ◽  
Activated Platelets ◽  
Platelet Membranes ◽  
Human Erythrocyte Membranes

This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

Effect of Integral Proteins on Frozen Membrane Fracture

1980 ◽  
Vol 38 ◽  
pp. 756-759 ◽  
Author(s):  
S. Kirchanski ◽  
D. Branton
Keyword(s):  
Lipid Bilayers ◽  
Freeze Fracture ◽  
Covalent Bond ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Experimental Protocol ◽  
Transmembrane Glycoprotein ◽  
Bond Breakage ◽  
Human Erythrocyte Membranes ◽  
Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

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