PHOTOCHEMICAL LABELING OF HUMAN ERYTHROCYTE-MEMBRANES WITH RADIOIODINATABLE AZIDOSALICYLIC ACID-DERIVATIVE OF GLOBOSIDE

In an attempt to define glycolipid functions we have prepared photoact ivatable, iodinatable derivative of globoside and used it for photoaff inity labeling of human erythrocyte membranes. Lysogloboside (Gb(4)Sph ) was prepared from globoside through deacylation in methanolic KOH fo llowed by re-N-acetylation of galactosaminyl residue. The NH2 group of sphingosine residue in Gb(4)Sph reacted with N-hydroxysuccinimidyl-4- azidosalicylic acid resulting in the formation of Gb(4)Sph-ASA which w as purified by preparative tie and column chromatography. It migrated on tie as a single spot in two solvent systems, was susceptible to lee ch ceramide glycanase and could be radioiodinated to a specific radioa ctivity of about 200 Ci/mmol. Gb(4)Sph-[I-125]ASA was incorporated int o human erythrocytes in a time and concentration-dependent manner. Bef ore photolysis 96% of the Gb(4)Sph-ASA could be removed with albumin b ut not with trypsin. After photolysis about 50% of the label was firml y bound to erythrocytes being resistant to albumin and trypsin treatme nt. The label was distributed between membrane proteins and lipids in about 1:2.3 ratio. Photolabeled proteins were analyzed by SDS-PAGE fol lowed by autoradiography and immunostaining. Most of the radioactivity was detected in band 3 and its proteolytic fragments irrespective of the duration of photolysis. Photolabeling of erythrocyte lipids was de monstrated by Sephadex LH-20 column chromatography.