Photoaffinity labeling of the human red-blood-cell urea-transporter polypeptide components : possible homology with the Kidd blood group antigen

• Published in: European journal of biochemistry Vol. 218; no. 2; pp. 447 - 455
• Main Authors: NEAU, P, DEGEILH, F, LAMOTTE, H, ROUSSEAU, B, RIPOCHE, P
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01.12.1993
• Subjects: Affinity Labels; Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Biological and medical sciences; Biological Transport; Erythrocyte Membrane - chemistry; Female; Fundamental and applied biological sciences. Psychology; Humans; Kidd Blood-Group System - chemistry; Membrane Proteins - chemistry; Phenylthiourea - analogs & derivatives; Phenylthiourea - chemistry; Proteins; Rana esculenta; Urea - metabolism; Urinary Bladder - metabolism; Antigen; Human; Blood group; Polypeptide; Kidd system; Red blood cell; Homology; Photoaffinity labelling; Carrier protein
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1993.tb18395.x

The tritiated urea analogue 1-(3-azido-4-chlorophenyl)-3methyl-2-thiourea ([3H]MeACPTU) was used as a probe to photolabel the human red-blood-cell membrane facilitated urea transporter. On irradiation, [3H]MeACPTU incorporated irreversibly into white ghost membranes. SDS/gel electrophoresis of membranes revealed radioactive incorporation in five major bands of 200, 110, 60, 40 and 14 kDa. The labeling of the 40-kDa and 60-kDa bands was partly prevented by the presence of a high concentration of other urea analogues such as thiourea and 1-(3,4-dichlorophenyl) 2-thiourea (DCPTU). The photolabeling pattern obtained with white ghosts of the Kidd blood-group type Jk(a-,b-) showed no labeling of the 40-kDa polypeptide. Protecting experiments carried out with anti-Jka, anti-Jkb and anti-Jk3 sera prevented radioactive incorporation in the 60-kDa band and in the 110-kDa band. Urea permeability of pink ghosts of blood type Jk(a+,b+) measured in the presence of Jk3 antibodies was 19% lower than the control values. However, urea permeability of frog urinary bladder epithelial cells was not affected by the presence of Jk-reactive antibodies. These results support the hypothesis that the Kidd antigen and the facilitated urea transporter are the same protein. Our estimation of the number of copies in each cell is close to that of the previously published value of 14000.