Photochemical labeling of HL-60 cell membrane proteins with radioiodinated, 4-azidosalicylic acid acylated derivatives of gangliosides
| Autor: | Grazyna Hoser, Mirosława Panasiewicz, Maciej Kawalec, Tadeusz Pacuszka |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Azides
Cell HL-60 Cells Photoaffinity Labels General Biochemistry Genetics and Molecular Biology Iodine Radioisotopes chemistry.chemical_compound Gangliosides medicine Humans Incubation Ganglioside Chromatography Molecular mass Chemistry Membrane Proteins Glycosphingolipid Trypsin Salicylates digestive system diseases Staining surgical procedures operative medicine.anatomical_structure Biochemistry Membrane protein lipids (amino acids peptides and proteins) medicine.drug |
| Zdroj: | Acta Biochimica Polonica. 45:403-415 |
| ISSN: | 1734-154X 0001-527X |
| DOI: | 10.18388/abp.1998_4235 |
| Popis: | To detect HL-60 human promyelocytic leukemia cell proteins involved in the uptake of gangliosides from the culture medium we used photoreactive, 4-azidosalicylic acid (ASA) acylated and radioiodinated (200 Ci/mmole) derivatives of GM3, GD3, GM1, and FucGM1 gangliosides. Gangliosides-ASA, added to the medium at 15-20 nM concentration, followed a similar time course of uptake. After 1 min incubation cell bound gangliosides-ASA could not be removed with trypsin, but only 5-10% remained after incubation with BSA. The proportion of cell bound gangliosides-ASA resistant to BSA treatment increased with time of incubation up to 76% after 20 h. As shown on TLC, GM3- and GD3-ASA were catabolized to LacSph-ASA and ceramide-ASA, while GM1-ASA was hydrolyzed to GM2-ASA. FucGM1-ASA was converted to GM1-ASA very slowly. Upon irradiation with UV lamp, cell bound gangliosides-ASA crosslinked to and photolabeled many proteins but the distribution of radioactivity after SDS/PAGE was very uneven and did not correlate with Coomassie staining. In all experiments the 42 kDa protein bands were most intensely photolabeled. Photolabeling of 42 kDa proteins decreased with time of incubation as compared to lower molecular mass pro teins. With all gangliosides-ASA used similar but not identical protein photolabeling patterns were obtained. Photolabeling patterns with GM3- and GD3-ASA differed from those with GM1- and FucGM1-ASA. |
| Databáze: | OpenAIRE |
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