Photochemical labeling of human erythrocyte membranes with radioiodinatable azidosalicylic acid derivative of globoside.
In an attempt to define glycolipid functions we have prepared photoactivatable, iodinatable derivative of globoside and used it for photoaffinity labeling of human erythrocyte membranes. Lysogloboside (Gb4Sph) was prepared from globoside through deacylation in methanolic KOH followed by re-N-acetylation of galactosaminyl residue. The NH2 group of sphingosine residue in Gb4 Sph reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid resulting in the formation of Gb4Sph-ASA which was purified by preparative tlc and column chromatography. It migrated on tlc as a single spot in two solvent systems, was susceptible to leech ceramide glycanase and could be radioiodinated to a specific radioactivity of about 200 Ci/mmol. Gb4Sph-[125I]ASA was incorporated into human erythrocytes in a time and concentration-dependent manner. Before photolysis 96% of the Gb4Sph-ASA could be removed with albumin but not with trypsin. After photolysis about 50% of the label was firmly bound to erythrocytes being resistant to albumin and trypsin treatment. The label was distributed between membrane proteins and lipids in about 1:2.3 ratio. Photolabeled proteins were analyzed by SDS-PAGE followed 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 demonstrated by Sephadex LH-20 column chromatography.
Pacuszka
T
T
Department of Biochemistry, Medical Center of Postgraduate Education, Warsaw, Poland.
Panasiewicz
M
M
D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis; D001345:Autoradiography; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005915:Globosides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D008563:Membrane Lipids / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D015394:Molecular Structure; D009844:Oligosaccharides / Q000032:analysis; D010782:Photolysis
D001457:Anion Exchange Protein 1, Erythrocyte; D005915:Globosides; D007457:Iodine Radioisotopes; D008563:Membrane Lipids; D008565:Membrane Proteins; D009844:Oligosaccharides; C095219:lysogloboside-4-azidosalicylamide; D006026:Glycoside Hydrolases; C051111:ceramide glycanase