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Photochemical labeling of HL-60 cell membrane proteins with radioiodinated, 4-azidosalicylic acid acylated derivatives of gangliosides

100%

Pacuszka T., Panasiewicz M., Hoser G., Kawalec M.

Acta Biochimica Polonica

1998

tom 45

nr 2

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 proteins. 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.

Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of GM3, GD3, GM1, and FucGM1 gangliosides

84%

Pacuszka T., Panasiewicz M.

Acta Biochimica Polonica

1998

tom 45

nr 2

Photoreactive gangliosides of high specific radioactivity may prove useful for studies on glycosphingolipid functions. We prepared 4-azidosalicylic acid (ASA) acylated derivatives of GM3, GD3, GM1, and FucGM1 gangliosides (gangliosides-ASA). Gangliosides-ASA were characterized by their TLC mobility, UV spectra, carbohydrate composition, and digestion with leech endoceramidase. After radioiodination to about 200 Ci/mmole gangliosides-ASA were used for photochemical labeling of human erythrocytes. Radioiodinated gangliosides-ASA were incorporated into erythrocytes in a time and concentration dependent manner, the kinetics and extent of incorporation being similar for all the gangliosides-ASA used. Radioiodinated gangliosides-ASA incorporated into erythrocytes were resistant to trypsin digestion while treatment with 1% BSA removed about 90% of the label. Incubation with cholera toxin protected radioiodinated GM1-ASA and, to a lesser extent, FucGM1-ASA but not GM3-ASA and GD3-ASA, against removal with BSA. After photolysis about 40-50% of radioactivity was firmly bound to erythrocyte lipids and proteins. The ratio of lipid- to protein-bound radioactivity ranged from 2.2:1 to 3.2:1. Photolabeled proteins were analyzed by SDS/PAGE followed by autoradiography. Band 3 was the most extensively photolabeled protein with all the radioiodinated gangliosides-ASA used. DIDS, an inhibitor of band 3 protein activity, caused reduction in photolabeling of this protein by about 20%.

Ograniczanie wyników

2 Acta Biochimica Polonica

2 Pacuszka T.

2 Panasiewicz M.

1 Hoser G.

1 Kawalec M.

2 1998

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Photochemical labeling of HL-60 cell membrane proteins with radioiodinated, 4-azidosalicylic acid acylated derivatives of gangliosides

Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of GM3, GD3, GM1, and FucGM1 gangliosides