Immunochemical studies of human red cell membrane proteins excreted in normal urine

Bron, Claude; Gallagher, Gabrielle

doi:10.1016/0019-2791(74)90074-3

Cited by 5 publications

(6 citation statements)

References 14 publications

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“…(c) Complement-Mediated Cytotoxicity. Cytotoxic tests with 51Cr-labeled erythrocytes from various species as targets were performed according to a modification of the technique previously described (Bron & Gallagher, 1974). In brief, 5 X 106 washed erythrocytes in 200 mL of Ringer's buffer containing 5% fetal calf serum (Ringer's) were incubated with 0.5 mL of Na251Cr2072' (1 mCi/mL) for 30 min at 30 °C for amphibian cells or 37 °C for mammal cells.…”

Section: Methodsmentioning

confidence: 99%

Immunochemical evidence for a transmembrane orientation of both the (sodium(1+), potassium(1+) ion-activated)-ATPase subunits

Girardet¹,

Geering²,

Frantes³

et al. 1981

Biochemistry

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Antibodies were raised against the large catalytic subunit (apparent Mr 96000) and the glycoprotein (apparent Mr 60000) of the sodium- and potassium-dependent adenosine triphosphatase [(Na+, K+)-ATPase] from Bufo marinus. The specificity of each antiserum was assessed by two-dimensional immunoelectrophoresis using toad kidney microsomes or the purified holoenzyme as a source of antigen and by indirect immunoprecipitation of detergent-solubilized (Na+, K+)-ATPase subunits from radioiodinated or biosynthetically labeled kidney holoenzyme, microsomes, or postnuclear supernatant. The anticatalytic subunit serum reacted exclusively with a 96000-dalton protein. The antiserum to the glycoprotein was rendered specific to this subunit by absorption with purified catalytic subunit. The two antisera were agglutinating and lytic in the presence of complement when toad erythrocytes were used as targets, indicating that antigenic determinants of both subunits were exposed on the cell surface. The specific reactivities with surface-exposed antigenic determinants of both subunits could be absorbed with toad red blood cells. Such absorbed antisera still reacted with detergent-treated or untreated kidney microsomes, revealing the presence of cytoplasmic and/or intramembranous antigenic sites. Our immunochemical data demonstrate that the glycoprotein subunit of (Na+, K+)-ATPase spans the lipid bilayer and confirm the transmembrane orientation of the catalytic subunit postulated from functional studies.

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Section: Methodsmentioning

confidence: 99%

Immunochemical evidence for a transmembrane orientation of both the (sodium(1+), potassium(1+) ion-activated)-ATPase subunits

Girardet¹,

Geering²,

Frantes³

et al. 1981

Biochemistry

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“…Rotational Diffusion Measurements. The flash photolysis technique used to measure band 3 rotation has previously been described in detail (Cherry et al, 1976; Nigg & Cherry, 1979a; Cherry, 1978). Briefly, band 3 was selectively labeled by incubating intact erythrocytes with the triplet probe eosin-5maleimide (Nigg & Cherry, 1979a).…”

Section: Methodsmentioning

confidence: 99%

“…All antisera were tested for the presence of antibodies directed to cell surface antigens. This was performed by complement-mediated hemolytic testing using 51Cr-labeled target cells as described previously (Bron & Gallagher, 1974).…”

Section: Methodsmentioning

confidence: 99%

“…Prior to flash photolysis experiments, ghosts were washed 3 times in the same buffer at 4 °C. The samples were then flushed with argon (Cherry, 1978), and flash photolysis measurements were made at 37 °C after a 10-min equilibration.…”

Section: Methodsmentioning

confidence: 99%

“…Band 3 and glycophorin A are the two major membranespanning proteins of the human erythrocyte membrane. Band 3 is a glycoprotein of molecular weight ~90 000 which comprises ~25% of the total membrane protein and is responsible for anion transport [for a review, see Cabantchik et al (1978)].…”

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Band 3-glycophorin A association in erythrocyte membranes demonstrated by combining protein diffusion measurements with antibody-induced cross-linking

Nigg¹,

Bron²,

Girardet³

et al. 1980

Biochemistry

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142

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A new approach to the study of molecular protein interactions in biological membranes is presented. The technique is based on measuring the rotation of a membrane protein in the presence and absence of specific antibodies directed toward a purported complex partner. As a first illustration of the method, the putative association of band 3 with glycophorin A in the human erythrocyte membrane was investigated. The rotational diffusion of band 3 was strongly reduced following cross-linking of glycophorin A with divalent antibodies. However, little or no effect on band 3 rotation was produced by monovalent antiglycophorin A Fab fragments, antispectrinor nonspecific antibodies, ruling out major effects on band 3 mobility due to steric hindrance, unspecific antibody adsorption, or transmembrane interactions involving spectrin. It is concluded that immobilization of band 3 by antiglycophorin A antibodies is directly caused by cross-linking of a preexisting band 3-glycophorin A complex in the human erythrocyte membrane.

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Immobilized Enzymes and Proteins in Urinalysis

Boguslaski¹,

Smith²

1977

Biomedical Applications of Immobilized Enzymes and Proteins

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Immunochemical studies of human red cell membrane proteins excreted in normal urine

Cited by 5 publications

References 14 publications

Immunochemical evidence for a transmembrane orientation of both the (sodium(1+), potassium(1+) ion-activated)-ATPase subunits

Immunochemical evidence for a transmembrane orientation of both the (sodium(1+), potassium(1+) ion-activated)-ATPase subunits

Band 3-glycophorin A association in erythrocyte membranes demonstrated by combining protein diffusion measurements with antibody-induced cross-linking

Immobilized Enzymes and Proteins in Urinalysis

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