Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody.
Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross-reactivity by inducing nonspecific protein aggregation.
Jhun
B H
BH
Biophysics Laboratory, Veterans Administration Medical Center, Buffalo, NY 14215.
Berenski
C J
CJ
Craik
J D
JD
Paterson
A R
AR
Cass
C E
CE
Jung
C Y
CY
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D003429:Cross Reactions; D003571:Cytochalasin B / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D000939:Epitopes; D004903:Erythrocyte Aggregation; D004912:Erythrocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism*; D008823:Micelles; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002352:Carrier Proteins; D003902:Detergents; D000939:Epitopes; D008565:Membrane Proteins; D008823:Micelles; D009004:Monosaccharide Transport Proteins; D033703:Nucleoside Transport Proteins; D003571:Cytochalasin B; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine